Aldehyde dehydrogenase variants and methods of use

ABSTRACT

The invention provides polypeptides and encoding nucleic acids of aldehyde dehydrogenase variants. The invention also provides cells expressing aldehyde dehydrogenase variants. The invention further provides methods for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells. The invention additional provides methods for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells.

This application claims the benefit of U.S. Provisional Application No. 62/480,194, filed Mar. 31, 2017, the entire contents of which are incorporated herein by reference.

Reference is made to the following provisional and international applications, which are incorporated herein by reference in their entireties: (1) U.S. Provisional Application No. 62/480,208 entitled “3-HYDROXYBUTYRYL-COA DEHYDROGENASE VARIANTS AND METHODS OF USE,” filed Mar. 31, 2017 (Attorney Docket No. 12956-409-888); (2) U.S. Provisional Application No. 62/480,270 entitled “PROCESS AND SYSTEMS FOR OBTAINING 1,3-BUTANEDIOL FROM FERMENTATION BROTHS,” filed Mar. 31, 2017 (Attorney Docket No. 12956-407-888); (3) International Patent Application No. ______ entitled “3-HYDROXYBUTYRYL-COA DEHYDROGENASE VARIANTS AND METHODS OF USE,” filed on even date herewith (Attorney Docket No. 12956-409-228); and (4) International Patent Application No. ______ entitled, “PROCESS AND SYSTEMS FOR OBTAINING 1,3-BUTANEDIOL FROM FERMENTATION BROTHS,” filed on even date herewith (Attorney Docket No. 12956-407-228).

BACKGROUND OF THE INVENTION

This application incorporates herein by reference a Sequence Listing as an ASCII text file entitled “12956-408-228_Sequence_Listing.txt” created on Mar. 27, 2018, and having a size of 498,061 bytes.

The present invention relates generally to organisms engineered to produce desired products, engineered enzymes that facilitate production of a desired product, and more specifically to enzymes and cells that produce desired products such as 3-hydroxybutyraldehyde, 1,3-butanediol, 4-hydroxybutyraldehyde, 1,4-butanediol, and related products and products derived therefrom.

Various commodity chemicals are used to make desired products for commercial use. Many of the commodity chemicals are derived from petroleum. Such commodity chemicals have various uses, including use as solvents, resins, polymer precursors, and specialty chemicals. Desired commodity chemicals include 4-carbon molecules such as 1,4-butanediol and 1,3-butanediol, upstream precursors and downstream products. It is desirable to develop methods for production of commodity chemicals to provide renewable sources for petroleum-based products and to provide less energy- and capital-intensive processes.

Thus, there exists a need for methods that facilitate production of desired products. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides polypeptides and encoding nucleic acids of aldehyde dehydrogenase variants. The invention also provides cells expressing aldehyde dehydrogenase variants. The invention further provides methods for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells. The invention additional provides methods for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary 1,3-butanediol (1,3-BDO) pathway that comprise an aldehyde dehydrogenase. FIG. 1 shows pathways from acetoacetyl-CoA to 1,3-butanediol. The enzymes are: (A) acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); (B) 3-oxobutyraldehyde reductase (ketone reducing); (C) 3-hydroxybutyraldehyde reductase, also referred to herein as 1,3-butanediol dehydrogenase; (D) acetoacetyl-CoA reductase (CoA-dependent, alcohol forming); (E) 3-oxobutyraldehyde reductase (aldehyde reducing); (F) 4-hydroxy, 2-butanone reductase; (G) acetoacetyl-CoA reductase (ketone reducing); (H) 3-hydroxybutyryl-CoA reductase (aldehyde forming), also referred to herein as 3-hydroxybutyraldehyde dehydrogenase; and (I) 3-hydroxybutyryl-CoA reductase (alcohol forming).

FIG. 2 shows an exemplary 1,4-butanediol (1,4-BDO) pathway that comprises an aldehyde dehydrogenase. Enzymes catalyzing the biosynthetic reactions are: (1) succinyl-CoA synthetase; (2) CoA-independent succinic semialdehyde dehydrogenase; (3) α-ketoglutarate dehydrogenase; (4) glutamate:succinate semialdehyde transaminase; (5) glutamate decarboxylase; (6) CoA-dependent succinic semialdehyde dehydrogenase; (7) 4-hydroxybutanoate dehydrogenase (also referred to as 4-hydroxybutyrate dehydrogenase); (8) α-ketoglutarate decarboxylase; (9) 4-hydroxybutyryl CoA:acetyl-CoA transferase; (10) butyrate kinase (also referred to as 4-hydroxybutyrate kinase); (11) phosphotransbutyrylase (also referred to as phospho-trans-4-hydroxybutyrylase); (12) aldehyde dehydrogenase (also referred to as 4-hydroxybutyryl-CoA reductase); (13) alcohol dehydrogenase (also referred to as 4-hydroxybutanal reductase or 4-hydroxybutyraldehyde reductase).

FIG. 3 shows a sequence alignment of ALD-1, ALD-2 and ALD-3. The sequences correspond to SEQ ID NOS:1, 2 and 3, respectively. Underlined in the figure are 2 loop regions, the first designated A, the second B, both involved in substrate specificity and enantiomer specificity as determined herein. Loop A in ALD-1 is sequence LQKNNETQEYSINKKWVGKD (SEQ ID NO:124), in ALD-2 is sequence IGPKGAPDRKFVGKD (SEQ ID NO:125), and in ALD-3 is sequence ITPKGLNRNCVGKD (SEQ ID NO:126). Loop B in ALD-1 is sequence SFAGVGYEAEGFTTFTIA (SEQ ID NO:127), in ALD-2 is sequence TYCGTGVATNGAHSGASALTIA (SEQ ID NO:128), and in ALD-3 is sequence SYAAIGFGGEGFCTFTIA (SEQ ID NO:129). The sequence and the length of the substrate specificity loop A and B from ALD-2 differ from those of ALD-1 and ALD-3; nevertheless the alignment shows sufficient conservation to facilitate identification of corresponding positions for substitution as described herein, and especially so if combined with 3D modeling as shown in FIG. 6. ALD-3 was used as the template for modeling of crystal structure; see FIG. 6 that shows the two loop regions interacting to affect substrate specificity and enantiomer specificity, especially when modified with exemplary substitutions as described herein. ALD-1 and ALD-3 are 51.9% identical. ALD-1 and ALD-2 are 35.9% identical. ALD-3 and ALD-2 are 40% identical. A consensus for Loop A based on alignment of ALD-1, ALD-2 and ALD-3 is IXPKG----XXNRKXVGKD (SEQ ID NO:5). A consensus for Loop B based on alignment of ALD-1, ALD-2 and ALD-3 is SYAGXGXXXE----GFXTFTIA (SEQ ID NO:6). It is understood that the specifically identified amino acids in the consensus sequences are conserved residues, whereas the positions marked with “X” are variable, and can correspond to any amino acid, as desired and disclosed herein. It is further understood that “----” can correspond to the presence or absence of a variable number of amino acid residues. An example of such a variable number of amino acid residues is shown in FIGS. 3 and 4A-4C. Further, it is understood that conserved residues in the consensus sequence can be substituted, for example, with conservative amino acids, as described herein (see, for example, FIGS. 4A-4C).

FIGS. 4A-4C show alignments of exemplary aldehyde deydrogenases (ALD), which representative alignments demonstrate identifying positions in ALDs that correspond to positions in the representative template ALD sequence where substitutions of the invention can be made. As in FIG. 3, underlined are 2 loop regions, the first designated A, the second B, both involved in substrate specificity and enantiomer specificity as determined herein. FIG. 4A shows an alignment of exemplary ALD sequences with a 40-55% cutoff compared to ALD-1. The sequences correspond to SEQ ID NOS: 1 (ALD-1), 13, 20 and 24 as indicated in FIG. 4A. FIG. 4B shows an alignment of exemplary ALD sequences with a 75-90% cutoff compared to ALD-1. The sequences correspond to SEQ ID NOS: 1 (ALD-1), 30, 33 and 37 as indicated in FIG. 4B. Loops A and B are underlined. FIG. 4C shows an alignment of exemplary ALD sequences with a 90% cutoff compared to ALD-1. The sequences correspond to SEQ ID NOS: 1 (ALD-1), 38, 40 and 44 as indicated in FIG. 4C. ALD-1 is 99%, 97%, and 95% identical to SEQ ID NOS: 38, 40 and 44, respectively. FIGS. 4A-4C demonstrate that corresponding positions for substitutions taught herein can be identified in ALDs that have at least 40% identity with ALD-1, especially the Loop A and B regions, and especially the very conserved Loop B region.

FIGS. 5A and 5B show enzyme activities of various exemplary aldehyde dehydrogenases. FIG. 5A shows the specific activity of ALD-2, ALD-1 and ALD-1 variants on 3 hydroxy-(R)-butyraldehyde (left bar in sets of bars) and 3 hydroxy-(S)-butyraldehyde (right bar in sets of bars). FIG. 5B shows the ratio of activity with the R to S form of 3-hydroxybutyraldehyde.

FIGS. 6A-6C show ribbon diagrams of the structure of the aldehyde dehydrogenase 959. The diagrams show docking of 3-hydroxy-(R)-butyraldehyde (FIG. 6A) or 3-hydroxy-(S)-butyraldehyde (FIG. 6B) into the structure of 959. FIG. 6C shows the same orientation as 3-hydroxy-(R)-butyraldehyde (R3HB).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to enzyme variants that have desirable properties and are useful for producing desired products. In a particular embodiment, the invention relates to aldehyde dehydrogenase variants, which are enzyme variants that have markedly different structural and/or functional characteristics compared to a wild type enzyme that occurs in nature. Thus, the aldehyde dehydrogenases of the invention or not naturally occurring enzymes. Such aldehyde dehydrogenase variants of the invention are useful in an engineered cell, such as a microbial organism, that has been engineered to produce a desired product. For example, as disclosed herein, a cell, such as a microbial organism, having a metabolic pathway can produce a desired product. An aldehyde dehydrogenase of the invention having desirable characteristics can be introduced into a cell, such as microbial organism, that has a metabolic pathway that uses an aldehyde dehydrogenase enzymatic activity to produce a desired product. Such aldehyde dehydrogenase variants are additionally useful as biocatalysts for carrying our desired reactions in vitro. Thus, the aldehyde dehydrogenase variants of the invention can be utilized in engineered cells, such as microbial organisms, to produce a desired product or as an in vitro biocatalyst to produce a desired product.

As used herein, the term “non-naturally occurring” when used in reference to a cell, a microbial organism or microorganism of the invention is intended to mean that the cell has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the cell's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a biosynthetic pathway for producing a desired product.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring cells can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to a cell or microbial organism is intended to mean a cell that is substantially free of at least one component as the referenced cell is found in nature, if such a cell is found in nature. The term includes a cell that is removed from some or all components as it is found in its natural environment. The term also includes a cell that is removed from some or all components as the cell is found in non-naturally occurring environments. Therefore, an isolated cell is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated cells include partially pure cells, substantially pure cells and cells cultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host cell. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the cell. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host cell. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a cell that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host cell on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a cell can be engineered to express two or more exogenous nucleic acids encoding a desired enzyme or protein, such as a pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host cell, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

As used herein, the term “gene disruption,” or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive or attenuated. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate or attenuate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non-naturally occurring cells of the invention. A gene disruption also includes a null mutation, which refers to a mutation within a gene or a region containing a gene that results in the gene not being transcribed into RNA and/or translated into a functional gene product. Such a null mutation can arise from many types of mutations including, for example, inactivating point mutations, deletion of a portion of a gene, entire gene deletions, or deletion of chromosomal segments.

As used herein, the term “growth-coupled” when used in reference to the production of a biochemical product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.

As used herein, the term “attenuate,” or grammatical equivalents thereof, is intended to mean to weaken, reduce or diminish the activity or amount of an enzyme or protein. Attenuation of the activity or amount of an enzyme or protein can mimic complete disruption if the attenuation causes the activity or amount to fall below a critical level required for a given function. However, the attenuation of the activity or amount of an enzyme or protein that mimics complete disruption, for example, complete disruption for one pathway, can still be sufficient for a separate pathway to continue to function. For example, attenuation of an endogenous enzyme or protein can be sufficient to mimic the complete disruption of the same enzyme or protein for production of a desired product of the invention, but the remaining activity or amount of enzyme or protein can still be sufficient to maintain other pathways, such as a pathway that is critical for the host cell to survive, reproduce or grow. Attenuation of an enzyme or protein can also be weakening, reducing or diminishing the activity or amount of the enzyme or protein in an amount that is sufficient to increase yield of a desired product of the invention, but does not necessarily mimic complete disruption of the enzyme or protein.

The non-naturally occurring cells of the invention can contain stable genetic alterations, which refers to cells that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host cell or organism such as E. coli and their corresponding metabolic reactions or a suitable source cell or organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring cell. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of Mycoplasma 5′-3′ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

Therefore, in identifying and constructing the non-naturally occurring cells of the invention having biosynthetic capability for a desired product, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced cell that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly for a gene disruption, evolutionally related genes can also be disrupted or deleted in a host cell to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.

Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch: −2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

In one embodiment, the invention provides an aldehyde dehydrogenase that is a variant of a wild type or parent aldehyde dehydrogenase. The aldehyde dehydrogenase of the invention converts an acyl-CoA to its corresponding aldehyde. Such an enzyme can also be referred to as an oxidoreductase that converts an acyl-CoA to its corresponding aldehyde. Such an aldehyde dehydrogenase of the invention can be classified as a reaction 1.2.1.b, oxidoreductase (acyl-CoA to aldehyde), where the first three digits correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity. Exemplary enzymatic conversions of an aldehyde dehydrogenase of the invention include, but are not limited to, the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde (also referred to as 3-HBal) (see FIG. 1), and the conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde (see FIG. 2). An aldehyde dehydrogenase of the invention can be used to produce desired products such as 3-hydroxybutyraldehyde (3-HBal), 1,3-butanediol (1,3-BDO), 4-hydroxybutyraldehyde (4-HBal), 1,4-butanediol (1,4-BDO), or other desired products such as a downstream product, including an ester or amide thereof, in a cell, such as a microbial organism, containing a suitable metabolic pathway, or in vitro. For example, 1,3-BDO can be reacted with an acid, either in vivo or in vitro, to convert to an ester using, for example, a lipase. Such esters can have nutraceutical, medical and food uses, and are advantaged when R-form of 1,3-butanediol is used since that is the form (compared to S-form or the racemic mixture that is made from petroleum or from ethanol by the acetaldehyde chemical synthesis route) best utilized by both animals and humans as an energy source (e.g., a ketone ester, such as (R)-3-hydroxybutyl-R-1,3-butanediol monoester (which has Generally Recognized As Safe (GRAS) approval in the United States) and (R)-3-hydroxybutyrate glycerol monoester or diester). The ketone esters can be delivered orally, and the ester releases R-1,3-butanediol that is used by the body (see, for example, WO2013150153). Thus the present invention is particularly useful to provide an improved enzymatic route and microorganism to provide an improved composition of 1,3-butanediol, namely R-1,3-butanediol, highly enriched or essentially enantiomerically pure, and further having improved purity qualities with respect to by-products.

1,3-Butanediol, also referred to as butylene glycol, has further food related uses including use directly as a food source, a food ingredient, a flavoring agent, a solvent or solubilizer for flavoring agents, a stabilizer, an emulsifier, and an anti-microbial agent and preservative. 1,3-Butanediol is used in the pharmaceutical industry as a parenteral drug solvent. 1,3-Butanediol finds use in cosmetics as an ingredient that is an emollient, a humectant, that prevents crystallization of insoluble ingredients, a solubilizer for less-water-soluble ingredients such as fragrances, and as an anti-microbial agent and preservative. For example, it can be used as a humectant, especially in hair sprays and setting lotions; it reduces loss of aromas from essential oils, preserves against spoilage by microorganisms, and is used as a solvent for benzoates. 1,3-Butanediol can be use at concentrations from 0.1 percent or less to 50 percent or greater. It is used in hair and bath products, eye and facial makeup, fragrances, personal cleanliness products, and shaving and skin care preparations (see, for example, the Cosmetic Ingredient Review board's report: “Final Report on the Safety Assessment of Butylene Glycol, Hexylene Glycol, Ethoxydiglycol, and Dipropylene Glycol”, Journal of the American College of Toxicology, Volume 4, Number 5, 1985, which is incorporated herein by reference). This report provides specific uses and concentrations of 1,3-butanediol (butylene glycol) in cosmetics; see for examples the report's Table 2 therein entitled “Product Formulation Data”.

In one embodiment, the invention provides an isolated nucleic acid molecule selected from: (a) a nucleic acid molecule encoding an amino acid sequence referenced as SEQ ID NO:1, 2 or 3 or in Table 4, wherein the amino acid sequence comprises one or more of the amino acid substitutions set forth in Table 1, 2 and/or 3; (b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes one or more of the amino acid substitutions set forth in Table 1, 2 and/or 3; (c) a nucleic acid molecule encoding an amino acid sequence comprising the consensus sequence of Loop A (SEQ ID NO:5) and/or Loop B (SEQ ID NO:6), wherein the amino acid sequence comprises one or more of the amino acid substitutions set forth in Table 1, 2 and/or 3; and (d) a nucleic acid molecule that is complementary to (a) or (b). In an embodiment, the amino acid sequence encoded by the nucleic acid molecule, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced in SEQ ID NO:1, 2 or 3 or in Table 4. The amino acid sequence can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or more, of the amino acid substitutions set forth in Table 1, 2 and/or 3, for example, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, i.e., up to all of the amino acid positions having a substitution.

The invention also provides a vector containing the nucleic acid molecule of the invention. In one embodiment, the vector is an expression vector. In one embodiment, the vector comprises double stranded DNA.

The invention also provides a nucleic acid encoding an aldehyde dehydrogenase polypeptide of the invention. A nucleic acid molecule encoding an aldehyde dehydrogenase of the invention can also include a nucleic acid molecule that hybridizes to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a nucleic acid molecule that can be used in the invention can be described as having a certain percent sequence identity to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number. For example, the nucleic acid molecule can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleic acid described herein.

Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleic acid sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65° C., for example, if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleic acid sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. The phrase low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5×Denhart's solution, 6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

A nucleic acid molecule encoding an aldehyde dehydrogenase of the invention can have at least a certain sequence identity to a nucleotide sequence disclosed herein. Accordingly, in some aspects of the invention, a nucleic acid molecule encoding an aldehyde dehydrogenase of the invention has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein by SEQ ID NO, GenBank and/or GI number.

Sequence identity (also known as homology or similarity) refers to sequence similarity between two nucleic acid molecules or between two polypeptides. Identity can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information (see also Altschul et al.,” J. Mol. Biol. 215:403410 (1990)).

In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule is a nucleic acid molecule encoding a variant of a reference polypeptide, wherein (i) the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, 2 or 3 or those in Table 4 (SEQ ID NOS:7-123), (ii) the variant comprises one or more amino acid substitutions relative to SEQ ID NO: 1, 2 or 3 or those in Table 4, and (iii) the one or more amino acid substitutions are selected from the amino acid substitutions shown in Tables 1-3. Tables 1-3 provide non-limiting lists of exemplary variants of SEQ ID NO: 1, 2 or 3 or those in Table 4. In one embodiment, for each variant in Tables 1-3, all positions except for the indicated position(s) are identical to SEQ ID NO: 1, 2 or 3 or those in Table 4. Amino acid substitutions are indicated by a letter indicating the identity of the original amino acid, followed by a number indicating the position of the substituted amino acid in SEQ ID NO: 1, 2 or 3 or those in Table 4, followed by a letter indicating the identity of the substituted amino acid. For example, “D12A” indicates that the aspartic acid at position 12 in SEQ ID NO: 1 or 2 is replaced with an alanine. The single-letter code used to identify amino acids is the standard code known by those skilled in the art. Some variants in Tables 1-3 comprise two or more substitutions, which is indicated by a list of substitutions. The one or more amino acid substitutions can be selected from any one of the variants listed in Tables 1-3, or from any combination of two or more variants listed in Tables 1-3. When selecting from a single variant in Tables 1-3, the resulting variant can comprise one or more of the substitutions of the selected variant in any combination, including all of the indicated substitutions or less than all of the indicated substitutions. When substitutions are selected from those of two or more variants in Tables 1-3, the resulting variant can comprise one or more of the substitutions of the selected variants, including all of the indicated substitutions or less than all of the indicated substitutions from each of the two or more selected variants, in any combination. For example, the resulting variant can comprise 1, 2, 3, or 4 substitutions from a single variant in Tables 1-3. As a further example, the resulting variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, or more substitutions selected from 1, 2, 3, 4, 5, or more selected variants of Tables 1-3. In some embodiments, the resulting variant comprises all of the indicated substitutions of a selected variant in Tables 1-3. In some embodiments, the resulting variant differs from SEQ ID NO: 1, 2 or 3 or those in Table 4 by at least one amino acid substitution, but less than 25, 20, 10, 5, 4, or 3 amino acid substitutions. In some embodiments, the resulting variant comprises, consists essentially of, or consists of a sequence as indicated by a variant selected from Tables 1-3, differing from SEQ ID NO: 1, 2 or 3 or those in Table 4 only at the indicated amino acid substitutions.

In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule encoding a variant of a reference polypeptide (the reference polypeptide having an amino acid sequence of SEQ ID NO: 1, 2 or 3 or those in Table 4), wherein the variant (i) comprises one or more amino acid substitutions of a corresponding variant selected from Table 1-3, and (ii) has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% 99%, or 100% sequence identity to the corresponding variant. In cases where the second variant has 100% sequence identity to the corresponding variant, the second variant comprises a sequence as indicated by a variant selected from Table 1-3, and may or may not have one or more additional amino acids at either or both the amino- and carboxy-termini. In some embodiments, the resulting variant has at least 80%, 85%, 90%, or 95% sequence identity to a corresponding variant selected from Table 1-3; in some cases, identity is at least 90% or more. In cases where the resulting variant is less than 100% identical to a corresponding variant selected from Table 1-3, the position of one or more of the amino acid substitutions indicated for the corresponding variant may shift (e.g. in the case of insertion or deletion of one or more amino acids), but still be contained within the resulting variant. For example, the aspartic acid to alanine substitution corresponding to “D12A” (at position 12 relative to SEQ ID NO: 1 or 2) may be present, but at a different position in the resulting variant. Whether an amino acid corresponds to an indicated substitution, albeit at a different position, can be determined by sequence alignment, as is well known in the art. In general, an alignment showing identity or similarity of amino acids flanking the substituted amino acid, such that the flanking sequences are considered to be aligned with a homologous sequence of another polypeptide, will allow the substituted amino acid to be positioned locally with respect to the corresponding variant of Table 1-3 to determine a corresponding position to make the substitution, albeit at a shifted numerical position in a given polypeptide chain. In one embodiment, a region comprising at least three to fifteen amino acids, including the substituted position, will locally align with the corresponding variant sequence with a relatively high percent identity, including at the position of the substituted amino acid along the corresponding variant sequence (e.g. 90%, 95%, or 100% identity). In some embodiments, the one or more amino acid substitutions (e.g. all or less than all of the amino acid substitutions) indicated by a corresponding variant selected from Table 1-3 is considered to be present in a given variant, even if occurring at a different physical position along a polypeptide chain, if the sequence of the polypeptide being compared aligns with the corresponding variant with an identical match or similar amino acid at the indicated position along the corresponding variant sequence when using a BLASTP alignment algorithm with default parameters, where a similar amino acid is one considered to have chemical properties sufficient for alignment with the variant position of interest using default parameters of the alignment algorithm.

In some embodiments, a nucleic acid molecule of the invention is complementary to a nucleic acid described in connection with any of the various embodiments herein.

It is understood that a nucleic acid of the invention or a polypeptide of the invention can exclude a wild type parental sequence, for example a parental sequence such as SEQ ID NOS: 1, 2 or 3 or sequences disclosed in Table 4. One skilled in the art will readily understand the meaning of a parental wild type sequence based on what is well known in the art. It is further understood that such a nucleic acid of the invention can exclude a nucleic acid sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, a polypeptide of the invention can exclude an amino acid sequence as found in nature. Thus, in a particular embodiment, the nucleic acid or polypeptide of the invention is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild type parental sequence or a naturally occurring amino acid sequence and/or that the nucleic acid sequence is not a wild type or naturally occurring nucleic acid sequence. A naturally occurring amino acid or nucleic acid sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleic acid or amino acid sequence that is not found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a nucleic acid and/or amino acid sequence of the invention. For example, a nucleic acid or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including variants as described herein, are included within the meaning of a nucleic acid or amino acid sequence of the invention that is not naturally occurring. An isolated nucleic acid molecule of the invention excludes a naturally occurring chromosome that contains the nucleic acid sequence, and can further exclude other molecules as found in a naturally occurring cell such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

Thus, an isolated nucleic acid sequence of the invention has physical and chemical differences compared to a naturally occurring nucleic acid sequence. An isolated or non-naturally occurring nucleic acid of the invention does not contain or does not necessarily have some or all of the chemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid sequence as found in nature. An isolated nucleic acid of the invention thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid sequence as found in a chromosome. A different chemical structure can occur, for example, by cleavage of phosphodiester bonds that release an isolated nucleic acid sequence from a naturally occurring chromosome. An isolated nucleic acid of the invention can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non-naturally occurring nucleic acid of the invention does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such as polymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non-naturally occurring nucleic acid of the invention also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid of the invention is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non-naturally occurring nucleic acids of the invention are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and/or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be an isolated or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that an isolated or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.

In one embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO:1, 2 or 3 or in Table 4, wherein the amino acid sequence comprises one or more of the amino acid substitutions set forth in Table 1, 2 and/or 3. In one embodiment, the invention provides an isolated polypeptide comprising the consensus amino acid sequence of Loop A (SEQ ID NO:5) and/or Loop B (SEQ ID NO:6).

In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO:1, 2 or 3 or in Table 4, wherein the amino acid sequence comprises one or more of the amino acid substitutions set forth in Table 1, 2 and/or 3, wherein the amino acid sequence, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acids sequence referenced as SEQ ID NO:1, 2 or 3 or in Table 4. In one embodiment, the amino acid sequence further comprises a conservative amino acid substitution in from 1 to 100 amino acid positions, wherein the positions are other than the one or more amino acid substitutions set forth in Table 1, 2 and/or 3. In another embodiment, the amino acid sequence comprises no modification at from 2 to 300 amino acid positions compared to the parent sequence, other than the one or more amino acid substitutions set forth in Table 1, 2 and/or 3, wherein the positions are selected from those that are identical to between 2, 3, 4, or 5 of the amino acid sequences referenced as SEQ ID NO:1, 2 or 3 or in Table 4. In one embodiment, the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or more, of the amino acid substitutions set forth in Table 1, 2 and/or 3, for example, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, i.e., up to all of the amino acid positions having a substitution.

In one embodiment, the polypeptide of the invention encodes an aldehyde dehydrogenase. In one embodiment, the polypeptide can convert 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde. In one embodiment, the polypeptide can convert 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. In one embodiment, the polypeptide has higher activity relative to the parental polypeptide. In one embodiment, the polypeptide has higher activity for 3-hydroxy-(R)-butyryl-CoA over 3-hydroxy-(S)-butyryl-CoA. In one embodiment, the polypeptide has higher specificity for 3-hydroxybutyryl-CoA over acetyl-CoA. In one embodiment, the polypeptide has higher specificity for 4-hydroxybutyryl-CoA over acetyl-CoA. In one embodiment, the polypeptide produces decreased byproducts in a cell or cell extract. In a particular embodiment, the byproduct is ethanol or 4-hydroxy-2-butanone. In one embodiment, the polypeptide has a higher kcat relative to the parental polypeptide.

In some embodiments, the invention provides an isolated polypeptide having an amino acid sequence disclosed herein, such SEQ ID NOS:1, 2 or 3 or those referenced in Table 4, wherein the amino acid sequence includes one or more variant amino acid positions as set forth in Tables 1, 2 and/or 3. In particular, such a polypeptide encodes an aldehyde dehydrogenase, which can convert an acyl-CoA to the corresponding aldehyde, for example, 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, or 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. In some aspects, the isolated polypeptide of the invention includes an amino acid sequence, other than the one or more variant amino acid positions as set forth in Tables 1, 2, and/or 3, with at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or is identical, to an amino acids sequence referenced as SEQ ID NOS:1, 2 or 3 or in Table 4. It is understood that a variant amino acid position can include any one of the 20 naturally occurring amino acids, a conservative substitution of a wild type or parental sequence at the corresponding position of the variant amino acid position, or a specific amino acid at the variant amino acid position such as those disclosed herein in Tables 1, 2 and/or 3. It is further understood that any of the variant amino acid positions can be combined to generate further variants. Variants with combinations of two or more variant amino acid positions exhibited activities greater than wild type. Thus, as exemplified herein, generating enzyme variants by combining active variant amino acid positions resulted in enzyme variants with improved properties. One skilled in the art can readily generate polypeptides with single variant positions or combinations of variant positions using methods well known to those skilled in the art to generate polypeptides with desired properties, including increased activity, increased specificity for the R form of 3-hydroxybutyryl-CoA or 3-hydroxybutyraldehyde over the S form, increased specificity for 3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA over acetyl-CoA, decreased byproduct formation, such as ethanol or 4-hydroxy-2-butanone, increased kcat, increased stability in vivo and/or in vitro and the like, as described herein.

“Homology” or “identity” or “similarity” refers to sequence similarity between two polypeptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polypeptide or polypeptide region (or a polynucleotide or polynucleotide region) has a certain percentage (for example, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of amino acids (or nucleotide bases) are the same in comparing the two sequences.

In certain embodiments, the invention provides an isolated polypeptide having an amino acid sequence that includes at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more variants in any combination disclosed herein. The variants can include any combination of the variants set forth in Tables 1, 2, and/or 3. In some embodiments, the isolated polypeptide is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, 2 or 3 or those in Table 4, and the polypeptide variant is selected from Table 1-3 and has one or more amino acid substitutions relative to SEQ ID NO: 1, 2 or 3 or those in Table 4.

In some embodiments, the isolated polypeptide is a variant of a reference polypeptide, wherein the reference polypeptide has an amino acid sequence of SEQ ID NO: 1, 2 or 3 or those in Table 4, the polypeptide variant comprises one or more amino acid substitutions relative to SEQ ID NO: 1, 2 or 3 or those in Table 4, where the one or more amino acid substitutions are selected from Table 1-3, and the polypeptide variant has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a corresponding variant selected from Table 1-3. The one or more amino acid substitutions can be selected from any one of the variants listed in Table 1-3, or from any combination of two or more variants listed in Table 1-3. When selecting from a single variant in Table 1-3, the resulting variant can comprise one or more of the substitutions of the selected variant in any combination, including all of the indicated substitutions or less than all of the indicated substitutions. When substitutions are selected from those of two or more variants in Table 1-3, the resulting variant can comprise one or more of the substitutions of the selected variants, including all of the indicated substitutions or less than all of the indicated substitutions from each of the two or more selected variants, in any combination. For example, the resulting variant can comprise 1, 2, 3, or 4 substitutions from a single variant in Table 1-3. As a further example, the resulting variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, or more substitutions selected from 1, 2, 3, 4, 5, or more selected variants of Table 1-3, including up to all positions being substituted, as disclosed herein. In some embodiments, the resulting variant comprises all of the indicated substitutions of a selected variant in Table 1-3. In some embodiments, the resulting variant differs from SEQ ID NO: 1, 2 or 3 or those in Table 4 by at least one amino acid substitution, but less than 25, 20, 10, 5, 4, or 3 amino acid substitutions. In some embodiments, the resulting variant comprises, consists essentially of, or consists of a sequence as indicated by a variant selected from Table 1-3, differing from SEQ ID NO: 1, 2 or 3 or those in Table 4 only at the indicated amino acid substitution(s).

In some embodiments, the resulting variant has at least 80%, 85%, 90%, or 95% sequence identity to a corresponding variant selected from Table 1-3; in some cases, identity is at least 90% or more. In cases where the resulting variant is less than 100% identical to a corresponding variant selected from Table 1-3, the position of one or more of the amino acid substitutions indicated for the corresponding variant may shift (e.g. in the case of insertion or deletion of one or more amino acids), but still be contained within the resulting variant. For example, the glycine to glutamic acid substitution corresponding to “D12A” (at position 12 relative to SEQ ID NO: 1 or 2) may be present, but at a different position in the resulting variant. Whether an amino acid corresponds to an indicated substitution, albeit at a different position, can be determined by sequence alignment, as described above and as well known in the art. In some embodiments, the one or more amino acid substitutions (e.g., all or less than all of the amino acid substitutions) indicated by a corresponding variant selected from Table 1-3 is considered to be present in a given variant, even if occurring at a different physical position along a polypeptide chain, if the sequence of the polypeptide being compared aligns with the corresponding variant with an identical match or similar amino acid at the indicated position along the corresponding variant sequence when using a BLASTP alignment algorithm with default parameters, where a similar amino acid is one considered to have chemical properties sufficient for alignment with the variant position of interest using default parameters of the alignment algorithm.

The variants alone or in combination can produce an enzyme that retains or improves the activity relative to a reference polypeptide, for example, the wild-type (native) enzyme. In some aspects, the polypeptide of the invention can have any combination of variants set forth in Tables 1, 2, and/or 3. In some aspects, the polypeptide of the invention having any combination of variants set forth in Tables 1, 2, and/or 3 can convert an acyl-CoA to the corresponding aldehyde, for example, 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, or 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. Methods of generating and assaying such polypeptides are well known to one of skill in the art.

In some embodiments, the isolated polypeptide of the invention can further include a conservative amino acid substitution in from 1 to 100 amino acid positions, or alternatively from 2 to 100 amino acid positions, or alternatively from 3 to 100 amino acid positions, or alternatively from 4 to 100 amino acid positions, or alternatively from 5 to 100 amino acid positions, or alternatively from 6 to 100 amino acid positions, or alternatively from 7 to 100 amino acid positions, or alternatively from 8 to 100 amino acid positions, or alternatively from 9 to 100 amino acid positions, or alternatively from 10 to 100 amino acid positions, or alternatively from 15 to 100 amino acid positions, or alternatively from 20 to 100 amino acid positions, or alternatively from 30 to 100 amino acid positions, or alternatively from 40 to 100 amino acid positions, or alternatively from 50 to 100 amino acid positions, or any integer therein, wherein the positions are other than the variant amino acid positions set forth in Tables 1, 2, and/or 3. In some aspects, the conservative amino acid sequence is a chemically conservative or an evolutionary conservative amino acid substitution. Methods of identifying conservative amino acids are well known to one of skill in the art, any one of which can be used to generate the isolated polypeptides of the invention.

In some embodiments, the isolated polypeptide of the invention can include no modification at from 2 to 300 amino acid positions, or alternatively 3 to 300 amino acid positions, or alternatively 4 to 300 amino acid positions, or alternatively 5 to 300 amino acid positions, or alternatively 10 to 300 amino acid positions, or alternatively 20 to 300 amino acid positions, or alternatively 30 to 300 amino acid positions, or alternatively 40 to 300 amino acid positions, or alternatively 50 to 300 amino acid positions, or alternatively 60 to 300 amino acid positions, or alternatively 80 to 300 amino acid positions, or alternatively 100 to 300 amino acid positions, or alternatively 150 to 300 amino acid positions, or alternatively 200 to 300 amino acid positions, or alternatively 250 to 300 amino acid positions, or any integer therein, compared to the parent (wild-type) sequence, wherein the positions are selected from those that are identical to between 2, 3, 4, or 5 of the amino acid sequences referenced as SEQ ID NOS:1, 2 or 3 or in Table 4.

It is understood that the variant polypeptides such as polypeptide variants of aldehyde dehydrogenase, as disclosed herein, can carry out a similar enzymatic reaction as the parent polypeptide, for example, converting an acyl-CoA to its corresponding aldehyde, such as converting 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, or converting 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. It is further understood that the polypeptide variants of the aldehyde dehydrogenase enzyme can include variants that provide a beneficial characteristic to the polypeptide, including but not limited to, increased activity, increased specificity for the R form of 3-hydroxybutyryl-CoA or 3-hydroxybutyraldehyde over the S form, increased specificity for 3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA over acetyl-CoA, decreased byproduct formation, such as ethanol or 4-hydroxy-2-butanone, increased kcat, increased stability in vivo and/or in vitro and the like (see Example). In a particular embodiment, the aldehyde dehydrogenase variant can exhibit an activity that is at least the same or higher than a wild type or parent polypeptide, that is, is higher than a parent polypeptide without the variant amino acid position(s). For example, the aldehyde dehydrogenase variants of the invention can have 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or even higher fold activity of the variant polypeptide over a wild type or parent polypeptide (see Example). It is understood that activity refers to the ability of an aldehyde dehydrogenase of the invention to convert a substrate to a product relative to a wild type or parent polypeptide under the same assay conditions.

In another particular embodiment, the aldehyde dehydrogenase variant can exhibit increased specificity for the R form of 3-hydroxybutyryl-CoA or 3-hydroxybutyraldehyde over the S form, for example, about 2 to 40 fold higher, for example, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10 or 2 to 5, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or even higher fold activity. Such an increased specificity can be measured, for example, by the ratio of activity for the R over the S form of 3-hydroxybutyryl-CoA or 3-hydroxybutyraldehyde.

In another particular embodiment, the aldehyde dehydrogenase variant can exhibit increased specificity for 3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA over acetyl-CoA, for example, 1.5 to 100, 1.5 to 95, 1.5 to 90, 1.5 to 85, 1.5 to 80, 1.5 to 75, 1.5 to 70, 1.5 to 65, 1.5 to 60, 1.5 to 55, 1.5 to 50, 1.5 to 45, 1.5 to 40, 1.5 to 35, 1.5 to 30, 1.5 to 25, 1.5 to 20, 1.5 to 15, 1.5 to 10, or 1.5 to 5, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold. Such an increased specificity can be measured, for example, by the ratio of activity for 3-hydroxybutyryl-CoA or 4-hydroxybutyryl-CoA over acetyl-CoA. Specificity is indicated by the activity on 3HB-CoA or 4HB-CoA divided by the activity on acetyl-CoA.

In another particular embodiment, the aldehyde dehydrogenase variant can exhibit decreased byproduct formation, such as ethanol and/or 4-hydroxy-2-butanone, for example, a decrease in byproduct formation of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. Such an aldehyde dehydrogenase variant can exhibit an activity that has decreased byproduct formation, as described above, relative to a wild type or a parent polypeptide, that is, a parent polypeptide without the variant amino acid position.

In another particular embodiment, the aldehyde dehydrogenase variant can exhibit increased kcat, for example, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10-fold or higher, relative to a wild type or a parent polypeptide, that is, a parent polypeptide without the variant amino acid position(s). The kcat is understood to refer to its well known meaning in enzymology of the turnover number, where kcat=Vmax/[ET], where Vmax is the rate of enzyme reaction with saturating substrate, and [ET] is the total enzyme concentration (see Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Kinetics, Wiley-Interscience, New York (1975)). Such an aldehyde dehydrogenase variant can exhibit an activity that has increased kcat relative to a wild type or a parent polypeptide, that is, a parent polypeptide without the variant amino acid position(s).

In another particular embodiment, the aldehyde dehydrogenase variant can exhibit increased stability, either in vitro or in vivo, or both, relative to a wild type or a parent polypeptide, that is, a parent polypeptide without the variant amino acid position(s). For example, the aldehyde dehydrogenase variant can exhibit increased stability in vitro in a cell lysate.

It is understood that, in certain embodiments, an aldehyde dehydrogenase variant can exhibit two or more of the characteristics described above, for example, two or more of the characteristics of (1) increased activity, (2) increased specificity for the R form of 3-hydroxybutyryl-CoA or 3-hydroxybutyraldehyde over the S form, (3) increased specificity for 3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA over acetyl-CoA, (4) decreased byproduct formation, such as ethanol and/or 4-hydroxy-2-butanone, (5) increased kcat, (6) increased stability in vivo and/or in vitro, and the like, in any combination. Such combinations include, for example, characteristics 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 2 and 3; 2 and 4; 2 and 5; 2 and 6; 3 and 4; 3 and 5; 3 and 6; 4 and 5; 4 and 6; 5 and 6; 1, 2 and 3; 1, 2 and 4; 1, 2 and 5; 1, 2 and 6; 1, 3 and 4; 1, 3 and 5; 1, 3 and 6; 1, 4 and 5; 1, 4 and 6; 1, 5 and 6; 2, 3 and 4; 2, 3 and 5; 2, 3 and 6; 2, 4 and 5; 2, 4 and 6; 2, 5 and 6; 3, 4 and 5; 3, 4 and 6; 3, 5 and 6; 4, 5 and 6; 1, 2, 3 and 4; 1, 2, 3 and 5; 1, 2, 3 and 6; 1, 2, 4 and 5; 1, 2, 4 and 6; 1, 2, 5 and 6; 1, 3, 4 and 5; 1, 3, 4 and 6; 1, 3, 5 and 6; 1, 4, 5 and 6; 2, 3, 4 and 5; 2, 3, 4 and 6; 2, 3, 5 and 6; 3, 4, 5 and 6; 1, 2, 3, 4 and 5; 1, 3, 4, 5 and 6; 1, 2, 4, 5 and 6; 1, 2, 3, 5 and 6; 1, 2, 3, 4 and 6; 2, 3, 4, 5 and 6; 1, 2, 3, 4, 5 and 6.

The polypeptides of the invention can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present invention can be obtained using well-known recombinant methods (see, for example, Sambrook et al., supra, 1989; Ausubel et al., supra, 1999). The methods and conditions for biochemical purification of a polypeptide of the invention can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

One non-limiting example of a method for preparing the invention polypeptide is to express nucleic acids encoding the polypeptide in a suitable host cell, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known purification methods, as described herein. Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors as described herein. Recombinantly expressed polypeptides of the invention can also be expressed as fusion proteins with appropriate affinity tags, such as glutathione S transferase (GST), poly His, streptavidin, and the like, and affinity purified, if desired. A polypeptide of the invention can retain the affinity tag, if desired, or optionally the affinity tag can be removed from the polypeptide using well known methods to remove an affinity tag, for example, using appropriate enzymatic or chemical cleavage. Thus, the invention provides polypeptides of the invention without or optionally with an affinity tag. In some embodiments, the invention provides a host cell expressing a polypeptide of the invention disclosed herein. An invention polypeptide can also be produced by chemical synthesis using a method of polypeptide synthesis well know to one of skill in the art (Merrifield, J. Am. Chem. Soc. 85:2149 (1964); Bodansky, M., Principles of Peptide Synthesis (Springer-Verlag, 1984); Houghten, Proc. Natl. Acad Sci., USA 82:5131 (1985); Grant Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. Principles of Peptide Synthesis. Springer-Verlag Inc., NY (1993)).

In some embodiments, the invention provides using a polypeptide disclosed herein as a biocatalyst. A “biocatalyst,” as used herein, refers to a biological substance that initiates or modifies the rate of a chemical reaction. A biocatalyst can be an enzyme. A polypeptide of the invention can be used to increase the rate of conversion of a substrate to a product as disclosed herein. In the context of an industrial reaction, a polypeptide of the invention can be used, absent a host cell expressing the polypeptide, to improve reactions generating 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, for example, using in vitro methods. In one embodiment, the invention provides use of the polypeptide of the invention as a biocatalyst.

In some embodiments of the invention, the polypeptide encoding an aldehyde dehydrogenase of the invention is provided as a cell lysate of a cell expressing the aldehyde dehydrogenase. In such a case, the cell lysate serves as a source of the aldehyde dehydrogenase for carrying out the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, or 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde, or the reverse reaction, in an in vitro reaction. In another embodiment, the aldehyde dehydrogenase can be provided in a partially purified form, for example, partially purified from a cell lysate. In another embodiment, the aldehyde dehydrogenase can be provided in substantially purified form, in which the aldehyde dehydrogenase is substantially purified from other components, such as the components of a cell extract. Methods for partially purifying or substantially purifying a polypeptide encoding an aldehyde dehydrogenase are well known in the art, as described herein. In some embodiments, the aldehyde dehydrogenase is immobilized to a solid support, for example, a bead, plate or membrane. In a particular embodiment, the aldehyde dehydrogenase comprises an affinity tag, and the affinity tag is used to immobilize the aldehyde dehydrogenase to a solid support. Such an affinity tag can include, but is not limited to, glutathione S transferase (GST), poly His, streptavidin, and the like, as described herein.

In some embodiments, the invention provides a composition having a polypeptide disclosed herein and at least one substrate for the polypeptide. Substrate for each of the polypeptides disclosed herein are described herein and are exemplified in the Figures. The polypeptide within the composition of the invention can react with a substrate under in vitro or in vivo conditions. In this context, an in vitro condition refers to a reaction in the absence of or outside of a cell, including a cell of the invention.

In one embodiment, the invention provides a composition comprising a polypeptide of the invention and at least one substrate for the polypeptide. In one embodiment, the polypeptide can react with the substrate under in vitro conditions. In one embodiment, the substrate is 3-hydroxybutyryl-CoA. In one embodiment, the substrate is 3-hydroxy-(R)-butyryl-CoA. In one embodiment, the substrate is 4-hydroxybutyryl-CoA.

In some embodiments, the invention provides a method of constructing a host strain that can include, among other steps, introducing a vector disclosed herein into a host cell, for example, that is capable of expressing an amino acid sequence encoded by the vector and/or is capable of fermentation. Vectors of the invention can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. Additional methods are disclosed herein, any one of which can be used in the method of the invention.

In an additional embodiment, the invention provides a cell that comprises a polypeptide of the invention, that is, an aldehyde dehydrogenase of the invention. Thus, the invention provides a non-naturally occurring cell comprising a polypeptide encoding an aldehyde dehydrogenase of the invention. Optionally, the cell can comprise a 3-HBal or 1,3-BDO pathway, or a 4-HBal or 1,4-BDO pathway, and additionally optionally include a pathway to produce a downstream product related thereto such as an ester or amide thereof. In some embodiments, the non-naturally occurring cell comprises at least one exogenous nucleic acid encoding an aldehyde dehydrogenase that converts an acyl-CoA to its corresponding aldehyde. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, in a particular embodiment, the invention provides a cell, in particular a non-naturally occurring cell, containing at least one exogenous nucleic acid encoding an aldehyde dehydrogenase, where the aldehyde dehydrogenase functions in a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, such as that shown in FIGS. 1 and 2.

In one embodiment, the invention provides a cell comprising a vector of the invention comprising a nucleic acid of the invention. The invention also provides a cell comprising a nucleic acid of the invention. In one embodiment, the nucleic acid molecule is integrated into a chromosome of the cell. In a particular embodiment, the integration is site-specific. In an embodiment of the invention, the nucleic acid molecule is expressed. In one embodiment, the invention provides a cell comprising a polypeptide of the invention.

In one embodiment, the cell comprising a vector, nucleic acid or polypeptide is a microbial organism. In a particular embodiment, the microbial organism is a bacterium, yeast or fungus. In a particular embodiment, the cell is an isolated eukaryotic cell.

In one embodiment, the cell comprises a pathway that produces 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof. In another embodiment, the cell comprises a pathway that produces 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof. In one embodiment, the cell is capable of fermentation. In one embodiment, the cell further comprises at least one substrate for the polypeptide of the invention expressed in the cell. In a particular embodiment, the substrate is 3-hydroxybutyryl-CoA. In a particular embodiment, the substrate is 3-hydroxy-(R)-butyryl-CoA. In one embodiment, the cell has higher activity for 3-hydroxy-(R)-butyryl-CoA over 3-hydroxy-(S)-butyryl-CoA. In another particular embodiment, the substrate is 4-hydroxybutyryl-CoA. The invention also provides culture medium comprising a cell of the invention.

The aldehyde dehydrogenase of the invention can be utilized in a pathway that converts an acyl-CoA to its corresponding aldehyde. Exemplary pathways for 3-HBal and/or 1,3-BDO that comprise an aldehyde dehydrogenase have been described, for example, in WO 2010/127319, WO 2013/036764, U.S. Pat. No. 9,017,983, US 2013/0066035, each of which is incorporated herein by reference.

Exemplary 3-HBal and/or 1,3-BDO pathways are shown in FIG. 1 and described in WO 2010/127319, WO 2013/036764, U.S. Pat. No. 9,017,983 and US 2013/0066035. Such a 3-HBal and/or 1,3-BDO pathway that comprises an aldehyde dehydrogenase includes, for example, (G) acetoacetyl-CoA reductase (ketone reducing); (H) 3-hydroxybutyryl-CoA reductase (aldehyde forming), also referred to herein as 3-hydroxybutyraldehyde dehydrogenase, an aldehyde dehydrogenase (ALD); and (C) 3-hydroxybutyraldehyde reductase, also referred to herein as a 1,3-BDO dehydrogenase (see FIG. 1). Acetoacetyl-CoA can be formed by converting two molecules of acetyl-CoA into one molecule of acetoacetyl-CoA employing a thiolase. Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into one molecule each of acetoacetyl-CoA and CoA (see WO 2013/036764 and US 2013/0066035).

An exemplary 1,3-BDO pathway is shown in FIG. 2 of WO 2010/127319. Briefly, acetoacetyl-CoA can be converted to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (ketone reducing) (EC 1.1.1.a) (step G of FIG. 1). 3-Hydroxybutyryl-CoA can be converted to 3-hydroxybutyraldehyde by 3-hydroxybutyryl-CoA reductase (aldehyde forming) (EC 1.2.1.b), also referred to herein as 3-hydroxybutyraldehyde dehydrogenase, including an aldehyde dehydrogenase of the invention (step H of FIG. 1). 3-Hydroxybutyraldehyde can be converted to 1,3-butanediol by 3-hydroxybutyraldehyde reductase (EC 1.1.1.a), also referred to herein as 1,3-BDO dehydrogenase (step C of FIG. 1).

As disclosed herein, aldehyde dehydrogenases of the invention can function in a pathway to convert 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde. In the pathway described above that comprises an aldehyde dehydrogenase that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, the pathway converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA (see FIG. 1). The aldehyde dehydrogenases of the invention can also be used in other 3-HBal and/or 1,3-BDO pathways that comprise 3-hydroxybutyryl-CoA as a substrate/product in the pathway. One skilled in the art can readily utilize an aldehyde dehydrogenase of the invention to convert 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde in any desired pathway that comprises such a reaction.

Exemplary 4-HBal and/or 1,4-BDO pathways are shown in FIG. 2 and described in WO 2008/115840, WO 2010/030711, WO 2010/141920, WO 2011/047101, WO 2013/184602, WO 2014/176514, U.S. Pat. Nos. 8,067,214, 7,858,350, 8,129,169, 8,377,666, US 2013/0029381, US 2014/0030779, US 2015/0148513 and US 2014/0371417. Such a 4-HBal and/or 1,4-BDO pathway that comprises an aldehyde dehydrogenase includes, for example, (1) succinyl-CoA synthetase; (2) CoA-independent succinic semialdehyde dehydrogenase; (3) α-ketoglutarate dehydrogenase; (4) glutamate:succinate semialdehyde transaminase; (5) glutamate decarboxylase; (6) CoA-dependent succinic semialdehyde dehydrogenase; (7) 4-hydroxybutanoate dehydrogenase; (8) α-ketoglutarate decarboxylase; (9) 4-hydroxybutyryl CoA:acetyl-CoA transferase; (10) butyrate kinase (also referred to as 4-hydroxybutyrate kinase); (11) phosphotransbutyrylase (also referred to as phospho-trans-4-hydroxybutyrylase); (12) aldehyde dehydrogenase (also referred to as 4-hydroxybutyryl-CoA reductase); (13) alcohol dehydrogenase, such as 1,4-butanediol dehydrogenase (also referred to as 4-hydroxybutanal reductase or 4-hydroxybutyraldehyde reductase) (see FIG. 2).

Similar to FIG. 2, exemplary 1,4-BDO pathways are shown in FIG. 8A of WO 2010/141920. Briefly, succinyl-CoA can be converted to succinic semialdehyde by succinyl-CoA reductase (or succinate semialdehyde dehydrogenase) (EC 1.2.1.b). Succinate semialdehyde can be converted to 4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a). Alternatively, succinyl-CoA can be converted to 4-hydroxybutyrate by succinyl-CoA reductase (alcohol forming) (EC 1.1.1.c). 4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), by 4-hydroxybutyryl-CoA hydrolase (EC 3.1.2.a) or by 4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). Alternatively, 4-hydroxybutyrate can be converted to 4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a). 4-Hydroxybutyryl-phosphate can be converted to 4-hydroxybutyryl-CoA by phosphotrans-4-hydroxybutyrylase (EC 2.3.1.a). Alternatively, 4-hydroxybutyryl-phosphate can be converted to 4-hydroxybutanal by 4-hydroxybutanal dehydrogenase (phosphorylating) (EC 1.2.1.d). 4-Hydroxybutyryl-CoA can be converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase) (EC 1.2.1.b), including by an aldehyde dehydrogenase variant of the invention. Alternatively, 4-hydroxybutyryl-CoA can be converted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c). 4-Hydroxybutanal can be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

Exemplary 1,4-BDO pathways are also shown in FIG. 8B of WO 2010/141920. Briefly, alpha-ketoglutarate can be converted to succinic semialdehyde by alpha-ketoglutarate decarboxylase (EC 4.1.1.a). Alternatively, alpha-ketoglutarate can be converted to glutamate by glutamate dehydrogenase (EC 1.4.1.a). 4-Aminobutyrate can be converted to succinic semialdehyde by 4-aminobutyrate oxidoreductase (deaminating) (EC 1.4.1.a) or 4-aminobutyrate transaminase (EC 2.6.1.a). Glutamate can be converted to 4-aminobutyrate by glutamate decarboxylase (EC 4.1.1.a). Succinate semialdehyde can be converted to 4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a). 4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), by 4-hydroxybutyryl-CoA hydrolase (EC 3.1.2.a), or by 4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). 4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a). 4-Hydroxybutyryl-phosphate can be converted to 4-hydroxybutyryl-CoA by phosphotrans-4-hydroxybutyrylase (EC 2.3.1.a). Alternatively, 4-hydroxybutyryl-phosphate can be converted to 4-hydroxybutanal by 4-hydroxybutanal dehydrogenase (phosphorylating) (EC 1.2.1.d). 4-Hydroxybutyryl-CoA can be converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase) (EC 1.2.1.b), including by an aldehyde dehydrogenase of the invention. 4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c). 4-Hydroxybutanal can be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

As disclosed herein, aldehyde dehydrogenases of the invention can function in a pathway to convert 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. In the pathways described above that comprise an aldehyde dehydrogenase that converts 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde, the pathways convert 4-hydroxybutyrate to 4-hydroxybutyryl-CoA or 4-hydroxybutyryl phosphate to 4-hydroxybutyryl-CoA (see FIG. 2). The aldehyde dehydrogenases of the invention can also be used in other 4-HBal and/or 1,4-BDO pathways that comprise 4-hydroxybutyryl-CoA as a substrate/product in the pathway. One skilled in the art can readily utilize an aldehyde dehydrogenase of the invention to convert 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde in any desired pathway that comprises such a reaction. For example, 4-oxobutyryl-CoA can be converted to 4-hydroxybutyryl-CoA as described and shown in WO 2010/141290, FIG. 9A. In addition, 5-hydroxy-2-oxopentanoic acid can be converted to 4-hydroxybutyryl-CoA as described and shown in WO 2010/141290, FIGS. 10 and 11. Also, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA and/or vinylacetyl-CoA can be converted to 4-hydroxybutyryl-CoA as described and shown in WO 2010/141290, FIG. 12. Additionally, 4-hydroxybut-2-enoyl-CoA can be converted to 4-hydroxybutyryl-CoA as described and shown in WO 2010/141290, FIG. 13. Thus, one skilled in the art will readily understand how to use an aldehyde dehydrogenase of the invention in a 4-HBal and/or 1,4-BDO pathway that comprises conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde, as desired.

Enzyme types required to convert common central metabolic intermediates into 1,3-BDO or 1,4-BDO are indicated above with representative Enzyme Commission (EC) numbers (see also WO 2010/127319, WO 2013/036764, WO 2008/115840, WO 2010/030711, WO 2010/141920, WO 2011/047101, WO 2013/184602, WO 2014/176514, U.S. Pat. Nos. 9,017,983, 8,067,214, 7,858,350, 8,129,169, 8,377,666, US 2013/0066035, US 2013/0029381, US 2014/0030779, US 2015/0148513, and US 2014/0371417). The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity. Exemplary enzymes include: 1.1.1.a, Oxidoreductase (ketone to hydroxyl or aldehyde to alcohol); 1.1.1.c, Oxidoreductase (2 step, acyl-CoA to alcohol); 1.2.1.b, Oxidoreductase (acyl-CoA to aldehyde); 1.2.1.c, Oxidoreductase (2-oxo acid to acyl-CoA, decarboxylation); 1.2.1.d, Oxidoreductase (phosphorylating/dephosphorylating); 1.3.1.a, Oxidoreductase operating on CH—CH donors; 1.4.1.a, Oxidoreductase operating on amino acids (deaminating); 2.3.1.a, Acyltransferase (transferring phosphate group); 2.6.1.a, Aminotransferase; 2.7.2.a, Phosphotransferase, carboxyl group acceptor; 2.8.3.a, Coenzyme-A transferase; 3.1.2.a, Thiolester hydrolase (CoA specific); 4.1.1.a, Carboxy-lyase; 4.2.1.a, Hydro-lyase; 4.3.1.a, Ammonia-lyase; 5.3.3.a, Isomerase; 5.4.3.a, Aminomutase; and 6.2.1.a, Acid-thiol ligase.

The aldehyde dehydrogenases of the invention can be utilized in a cell or in vitro to convert an acyl-CoA to its corresponding aldehyde. As disclosed herein, the aldehyde dehydrogenases of the invention have beneficial and useful properties, including but not limited to increased specificity for the R enantiomer of 3-hydroxybutyryl-CoA over the S enantiomer, increased specificity for 3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA over acetyl-CoA, increased activity, decreased byproduct production, increased kcat, and the like. Aldehyde dehydrogenases of the invention can be used to produce the R-form of 1,3-butanediol (also referred to as (R)-1,3-butanediol), by enzymatically converting the product of an aldehyde dehydrogenase of the invention, 3-hydroxy-(R)-butyraldehyde, to (R)-1,3-butanediol using a 1,3-butanediol dehydrogenase.

The bio-derived R-form of 1,3-butanediol can be utilized for production of downstream products for which the R-form is preferred. In some embodiments, the R-form can be utilized as a pharmaceutical and/or nutraceutical (see WO 2014/190251). For example, (R)-1,3-butanediol can be used to produce (3R)-hydroxybutyl (3R)-hydroxybutyrate, which can have beneficial effects such as increasing the level of ketone bodies in the blood. Increasing the level of ketone bodies can lead to various clinical benefits, including an enhancement of physical and cognitive performance and treatment of cardiovascular conditions, diabetes and treatment of mitochondrial dysfunction disorders and in treating muscle fatigue and impairment (see WO 2014/190251). The bio-derived R-form of 1,3-butanediol can be utilized for production of downstream products in which a non-petroleum based product is desired, for example, by substituting petroleum-derived racemate 1,3-butanediol, its S-form or its R-form, with the bio-derived R-form.

In one embodiment, the invention provides 3-HBal or 1,3-BDO, or downstream products related thereto, such as an ester or amide thereof, enantiomerically enriched for the R form of the compound. In some embodiments, the 3-HBal or 1,3-BDO is a racemate enriched in R-enantiomer, that is, includes more R-enantiomer than S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 55% or more R-enantiomer and 45% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 60% or more R-enantiomer and 40% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 65% or more R-enantiomer and 35% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 70% or more R-enantiomer and 30% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 75% or more R-enantiomer and 25% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 80% or more R-enantiomer and 20% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 85% or more R-enantiomer and 15% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 90% or more R-enantiomer and 10% or less S-enantiomer. For example, the 3-HBal or 1,3-BDO racemate can include 95% or more R-enantiomer and 5% or less S-enantiomer. In some embodiments, the 3-HBal or 1,3-BDO, or downstream products related thereto such as an ester or amide thereof, is greater than 90% R form, for example, greater than 95%, 96%, 97%, 98%, 99% or 99.9% R form. In one embodiment, the 3-HBal and/or 1,3-BDO, or downstream products related thereto, such as an ester or amide thereof, is ≥55% R-enantiomer, ≥60% R-enantiomer, ≥65% R-enantiomer, ≥70% R-enantiomer, ≥75% R-enantiomer, ≥80% R-enantiomer, ≥85% R-enantiomer, ≥90% R-enantiomer, or ≥95% R-enantiomer, and can be highly chemically pure, e.g., ≥99%, for example, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8% or ≥99.9% R-enantiomer.

In one embodiment, a petroleum-derived racemic mixture of a precursor of 3-HBal and/or 1,3-BDO, in particular a racemic mixture of 3-hydroxybutyryl-CoA, is used as a substrate for an aldehyde dehydrogenase of the invention, which exhibits increased specificity for the R form over the S form, to produce 3-HBal or 1,3-BDO, or a downstream product related thereto such as an ester or amide thereof, that is enantiomerically enriched for the R form. Such a reaction can be carried out by feeding a petroleum-derived precursor to a cell that expresses an aldehyde dehydrogenase of the invention, in particular a cell that can convert the precursor to 3-hydroxybutyryl-CoA, or can be carried out in vitro using one or more enzymes to convert the petroleum-derived precursor to 3-hydroxybutyryl-CoA, or a combination of in vivo and in vitro reactions. A reaction to produce 4-hydroxybutyryl-CoA with an aldehyde dehydrogenase of the invention can similarly be carried out by feeding a petroleum-derived precursor to a cell that expresses an aldehyde dehydrogenase of the invention, in particular a cell that can convert the precursor to 4-hydroxybutyryl-CoA, or can be carried out in vitro using one or more enzymes to convert the petroleum-derived precursor to 4-hydroxybutyryl-CoA, or a combination of in vivo and in vitro reactions.

While generally described herein as a cell that contains a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway comprising an aldehyde dehydrogenase of the invention, it is understood that the invention also provides a cell comprising at least one exogenous nucleic acid encoding an aldehyde dehydrogenase of the invention. The aldehyde dehydrogenase can be expressed in a sufficient amount to produce a desired product, such a product of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or a downstream product related thereto such as an ester or amide thereof. Exemplary 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathways are shown in FIGS. 1 and 2 and are described herein.

It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of FIGS. 1 and 2, can be utilized to generate a cell that produces any pathway intermediate or product, as desired, in particular a pathway that utilizes an aldehyde dehydrogenase of the invention. As disclosed herein, such a cell that produces an intermediate can be used in combination with another cell expressing one or more upstream or downstream pathway enzymes to produce a desired product. However, it is understood that a cell that produces a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate can be utilized to produce the intermediate as a desired product.

The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

As disclosed herein, a product or pathway intermediate that is a carboxylic acid can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix “-ate,” or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that carboxylate products or intermediates includes ester forms of carboxylate products or pathway intermediates, such as O-carboxylate and S-carboxylate esters. O- and S-carboxylates can include lower alkyl, that is C1 to C6, branched or straight chain carboxylates. Some such O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- or S-carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- or S-carboxylates. O-carboxylates can be the product of a biosynthetic pathway. Other biosynthetically accessible O-carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such as heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations. O-carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an O- or S-carboxylate. S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters.

The cells of the invention can be produced by introducing an expressible nucleic acid encoding an aldehyde dehydrogenase of the invention, and optionally expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathways, and further optionally a nucleic acid encoding an enzyme that produces a downstream product related to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO such as an ester or amide thereof. Depending on the host cell chosen, nucleic acids for some or all of a particular 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway, or downstream product, can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is included for the deficient enzyme(s) or protein(s) to achieve 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthesis, or exogenous expression of endogenously expressed genes can be provided to increase expression of pathway enzymes, if desired. Thus, a cell of the invention can be produced by introducing an aldehyde dehydrogenase of the invention, and optionally exogenous enzyme or protein activities to obtain a desired biosynthetic pathway, or by introducing one or more exogenous enzyme or protein activities, including an aldehyde dehydrogenase of the invention that, together with one or more endogenous enzymes or proteins, produces a desired product such as 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof.

Host cells can be selected from, and the non-naturally cells expressing an aldehyde dehydrogenase of the invention generated in, for example, bacteria, yeast, fungus or any of a variety of microorganisms applicable or suitable to fermentation processes. Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobiospirillum; the order Pasteurellales, family Pasteurellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas. Non-limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium eth, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.

Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, and the like. A particularly useful host organism that is a yeast includes Saccharomyces cerevisiae.

Although generally described herein as utilizing a cell that is a microbial organism as a host cell, particularly for producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, it is understood that a host cell can be a cell line of a higher eukaryote, such as a mammalian cell line or insect cell line. Thus, it is understood that reference herein to a host cell that is a microbial organism can alternatively utilize a higher eukaryotic cell line to produce a desired product. Exemplary higher eukaryotic cell lines include, but are not limited to, Chinese hamster ovary (CHO), human (Hela, Human Embryonic Kidney (HEK) 293, Jurkat), mouse (3T3), primate (Vero), insect (Sf9), and the like. Such cell lines are commercially available (see, for example, the American Type Culture Collection (ATCC; Manassas Va.); Life Technologies, Carlsbad Calif.). It is understood that any suitable host cell can be used to introduce an aldehyde dehydrogenase of the invention, and optionally metabolic and/or genetic modifications to produce a desired product.

Depending on the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway constituents of a selected host cell, the non-naturally occurring cells of the invention will include at least one exogenously expressed 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathways, or a downstream product related thereto such as an ester or amide thereof, including an aldehyde dehydrogenase of the invention. For example, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid, including an aldehyde dehydrogenase of the invention. In a host deficient in all enzymes or proteins of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or a downstream product related thereto such as an ester or amide thereof, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or a downstream product related thereto such as an ester or amide thereof, can be included, including an aldehyde dehydrogenase of the invention.

Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway deficiencies of the selected host cell if a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway is to be included in the cell. Therefore, a non-naturally occurring cell of the invention can have one, two, three, four, five, six, seven, eight, and so forth, depending on the particular pathway, up to all nucleic acids encoding the enzymes or proteins constituting a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring cells also can include other genetic modifications that facilitate or optimize 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthesis or that confer other useful functions onto the host cell. One such other functionality can include, for example, augmentation of the synthesis of one or more of the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway precursors such acetyl-CoA or acetoacetyl-CoA.

Generally, a host cell is selected such that it can express an aldehyde dehydrogenase of the invention, and optionally produces the precursor of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, in a cell containing such a pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host cell. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a cell that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or a downstream product related thereto such as an ester or amide thereof, if desired.

In some embodiments, a non-naturally occurring cell of the invention is generated from a host that contains the enzymatic capability to synthesize 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. In this specific embodiment it can be useful to increase the synthesis or accumulation of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway product to, for example, drive 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway reactions toward 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO production, or a downstream product related thereto such as an ester or amide thereof. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway enzymes or proteins, including an aldehyde dehydrogenase of the invention. Overexpression of the enzyme or enzymes and/or protein or proteins of the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes, including exogenous expression of an aldehyde dehydrogenase of the invention. Therefore, naturally occurring organisms can be readily converted to non-naturally occurring cells of the invention, for example, producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO or a downstream product related thereto such as an ester or amide thereof, through overexpression of one, two, three, four, five, six, seven, eight, or more, depending on the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, that is, up to all nucleic acids encoding 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway enzymes or proteins, or enzymes that produce a downstream product related thereto such as an ester or amide thereof. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway, or a downstream product related thereto such as an ester or amide thereof.

In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring cell.

It is understood that any of the one or more exogenous nucleic acids can be introduced into a cell to produce a non-naturally occurring cell of the invention. The nucleic acids can be introduced so as to confer, for example, a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, biosynthetic pathway onto the cell, including introducing a nucleic acid encoding an aldehyde dehydrogenase of the invention. Alternatively, encoding nucleic acids can be introduced to produce a cell having the biosynthetic capability to catalyze some of the required reactions to confer 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic capability to produce an intermediate. For example, a non-naturally occurring cell having a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, including an aldehyde dehydrogenase of the invention. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell of the invention, including an aldehyde dehydrogenase of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring cell of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

In addition to the biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, as described herein, the non-naturally occurring cells and methods of the invention also can be utilized in various combinations with each other and/or with other cells and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO other than use of the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO producers is through addition of another cell capable of converting a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO. One such procedure includes, for example, the fermentation of a cell that produces a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate. The 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate can then be used as a substrate for a second cell that converts the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO. The 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate can be added directly to another culture of the second organism or the original culture of the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate producers can be depleted of these cells by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. A cell that produces a downstream product related to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO such as an ester or amide thereof, can optionally be included to produce such a downstream product.

Alternatively, such enzymatic conversions can be carried out in vitro, with a combination of enzymes or sequential exposure of substrates to enzymes that result in conversion of a substrate to a desired product. As another alternative, a combination of cell-based conversions and in vitro enzymatic conversions can be used, if desired.

In other embodiments, the non-naturally occurring cells and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO or a downstream product related thereto such as an ester or amide thereof. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different cells, and the different cells can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one cell is the substrate for a second cell until the final product is synthesized. For example, the biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be accomplished by constructing a cell that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO also can be biosynthetically produced from cells through co-culture or co-fermentation using two different cells in the same vessel, where the first cell produces a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO intermediate and the second cell converts the intermediate to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof.

Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring cells and methods of the invention together with other cells, with the co-culture of other non-naturally occurring cells having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof.

Sources of encoding nucleic acids for a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway enzyme or protein, or a downstream product related thereto such as an ester or amide thereof, can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia col, Saccharomyces cerevisiae, Saccharomyces kluyveri, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beierinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandi, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzu, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodani, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate-producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loft, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M Tsukamurella paurometabola D SM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Aquifex aeolicus, Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus niger, Aspergillus terreus, Bacillus subtilis, Bos Taurus, Candida albicans, Candida tropicalis, Chlamydomonas reinhardtii, Chlorobium tepidum, Citrobacter koseri, Citrus junos, Clostridium acetobutylicum, Clostridium kluyveri, Clostridium saccharoperbutylacetonicum, Cyanobium PCC7001, Desulfatibacillum alkenivorans, Dictyostehium discoideum, Fusobacterium nucleatum, Haloarcula marismortui, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumoniae, Kluyveromyces lactis, Lactobacillus brevis, Leuconostoc mesenteroides, Metallosphaera sedula, Methanothermobacter thermautotrophicus, Mus musculus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium smegmatis, Nicotiana tabacum, Nocardia iowensis, Oryctolagus cuniculus, Penicillium chrysogenum, Pichia pastoris, Porphyromonas gingivalis, Porphyromonas gingivais, Pseudomonas aeruginos, Pseudomonas putida, Pyrobaculum aerophilum, Ralstonia eutropha, Rattus norvegicus, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Salmonella enteric, Salmonella typhimurium, Schizosaccharomyces pombe, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodan, Thermoanaerobacter tengcongensis, Thermus thermophilus, Trypanosoma brucei, Tsukamurella paurometabola, Yarrowia lipolytica, Zoogloea ramigera and Zymomonas mobilis, Clostridium species, including but no limited to Clostridium saccharoperbutylacetonicum, Clostridium beierinckii, Clostridium saccharobutylicum, Clostridium botulinum, Clostridium methylpentosum, Clostridium sticklandu, Clostridium phytofermentans, Clostridium saccharolyticum, Clostridium asparagiforme, Clostridium celatum, Clostridium carboxidivorans, Clostridium clostridioforme, Clostridium boteae, Caldalkalibacillus thermarum, Clostridium botulinum, Pelosinus fermentans, Thermoanaerobacterium thermosaccharolyticum, Desulfosporosinus speices, Thermoanaerobacterium species, including but not limited to Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium xylanolyticum, Acetonema longum, Geobacillus species, including but not limited to Geobacillus thermoglucosidans, Bacillus azotoformans, Thermincolapotens, Fusobacterium species, including but not limited to Fusobacterium nucleatum, Fusobacterium ulcerans, Fusobacterium varium, Ruminococcus species, including but not limited to Ruminococcus gnavus, Ruminococcus obeum, Lachnospiraceae bacterium, Flavonifractor plautii, Roseburia inulinivorans, Acetobacterium woodii, Eubacterium species, including but not limited to Eubacterium plexicaudatum, Eubacterium hallii, Eubacterium limosum, Eubacterium yurii, Eubacteriaceae bacterium, Thermosediminibacter oceani, Ilyobacter polytropus, Shuttleworthia satelles, Halanaerobium sacchcrolyticum, Thermoanaerobacter ethanolicus, Rhodospirillum rubrum, Vibrio, Propionibacterium propionicum as well as other exemplary species disclosed herein or available as source organisms for corresponding genes, including the source organisms of the aldehyde dehydrogenases described in Table 4. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, including expression of an aldehyde dehydrogenase of the invention, described herein with reference to a particular organism such as E. coli can be readily applied to other cells such as microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

In some instances, such as when an alternative 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway exists in an unrelated species, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all cells using the cognate metabolic alterations to those exemplified herein to construct a cell in a species of interest that will synthesize 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, if desired, including introducing an aldehyde dehydrogenase of the invention.

Methods for constructing and testing the expression levels of a non-naturally occurring host producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, including an aldehyde dehydrogenase of the invention, can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

An exogenous nucleic acid encoding an aldehyde dehydrogenase of the invention, and optionally exogenous nucleic acid sequences involved in a pathway for production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E coli (Hoffieister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include a nucleic acid encoding an aldehyde dehydrogenase of the invention, and/or optionally one or more 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO biosynthetic pathway encoding nucleic acids, or nucleic acids encoding an enzyme that produces a downstream product related to 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO such as an ester or amide thereof, as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the host cells of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences encoding an aldehyde dehydrogenase of the invention or encoding polypeptides involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

A vector or expression vector can also be used to express an encoded nucleic acid to produce an encoded polypeptide by in vitro transcription and translation. Such a vector or expression vector will comprise at least a promoter, and includes the vectors described herein above. Such a vector for in vitro transcription and translation generally is double stranded DNA. Methods of in vitro transcription and translation are well known to those skilled in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999)). Kits for in vitro transcription and translation are also commercially available (see, for example, Promega, Madison, Wis.; New England Biolabs, Ipswich, Mass.; Thermo Fisher Scientific, Carlsbad, Calif.).

In one embodiment, the invention provides a method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising culturing a cell of the invention to produce 3-HBal and/or 1,3-BDO, or an ester or amide thereof. Such a cell expresses a polypeptide of the invention. In one embodiment, the invention provides a method for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing a cell of the invention to produce 4-HBal and/or 1,4-BDO, or an ester or amide thereof. In one embodiment, the cell is in a substantially anaerobic culture medium. In one embodiment, the method can further comprise isolating or purifying the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, or ester or amide thereof. In a particular embodiment, the isolating or purifying comprises distillation.

In one embodiment, the invention provides a process for producing a product of the invention, comprising chemically reacting the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, with itself or another compound in a reaction that produces the product.

In one embodiment, the invention provides a method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising providing a substrate to a polypeptide of the invention and converting the substrate to 3-HBal and/or 1,3-BDO, wherein the substrate is a racemic mixture of 1,3-hydroxybutyryl-CoA. In one embodiment, the 3-HBal and/or 1,3-BDO is enantiomerically enriched for the R form. In one embodiment, the invention provides a method for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising providing a substrate to a polypeptide of the invention and converting the substrate to 4-HBal and/or 1,4-BDO, wherein the substrate is 1,4-hydroxybutyryl-CoA. In one embodiment, the polypeptide is present in a cell, in a cell lysate, or is isolated from a cell or cell lysate.

In one embodiment, the invention provides a method for producing 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO, comprising incubating a lysate of a cell of the invention to produce 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO. In one embodiment, the cell lysate is mixed with a second cell lysate, wherein the second cell lysate comprises an enzymatic activity to produce a substrate of a polypeptide of the invention, or a downstream product of 3-HBal and/or 1,3-BDO. or 4-HBal and/or 1,4-BDO.

The invention also provides a method for producing a polypeptide of the invention, comprising expressing the polypeptide in a cell. The invention additionally provides a method for producing a polypeptide of the invention, comprising in vitro transcribing and translating a nucleic acid of the invention or a vector of the invention to produce the polypeptide.

As described herein, a cell can be used to express an aldehyde dehydrogenase of the invention, and optionally the cell can include a metabolic pathway that utilizes an aldehyde dehydrogenase of the invention to produce a desired product, such as 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO. Such methods for expressing a desired product are described herein. Alternatively, an aldehyde dehydrogenase of the invention can be expressed, and/or a desired product produced, in a cell lysate, for example, a cell lysate of a cell expressing an aldehyde dehydrogenase of the invention, or a cell expressing an aldehyde dehydrogenase of the invention and a metabolic pathway to produce a desired product, as described herein. In another embodiment, an aldehyde dehydrogenase of the invention can be expressed by in vitro transcription and translation, in which the aldehyde dehydrogenase is produced in a cell free system. The aldehyde dehydrogenase expressed by in vitro transcription and translation can be used to carry out a reaction in vitro. Optionally, other enzymes, or cell lysate(s) containing such enzymes, can be used to convert the product of the aldehyde dehydrogenase enzymatic reaction to a desired downstream product in vitro.

Suitable purification and/or assays to test for the expression of an aldehyde dehydrogenase, or for production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, including assays to test for aldehyde dehydrogenase activity, can be performed using well known methods (see also Example). Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art (see also Example).

The 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or other desired product, such as a downstream product related thereto such as an ester or amide thereof, can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring cells expressing an aldehyde dehydrogenase of the invention described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the cells that produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be cultured for the biosynthetic production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. Accordingly, in some embodiments, the invention provides culture medium containing the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate described herein. In some aspects, the culture medium can also be separated from the non-naturally occurring cells of the invention that produced the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate. Methods for separating a cell from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.

For the production of an aldehyde dehydrogenase of the invention, or of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, in a cell expressing an aldehyde dehydrogenase of the invention, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United States publication 2009/0047719, filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high yields of a desired product such as 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring cell. Such sources include, for example: sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, and it is understood that a carbon source can be used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the cells of the invention for the expression of an aldehyde dehydrogenase of the invention, and optionally production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product thereof, such as an ester or amide thereof.

In addition to renewable feedstocks such as those exemplified above, the cells of the invention that produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO or a downstream product thereof, such as an ester or amide thereof, also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H₂ and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H₂ and CO, syngas can also include CO₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO₂ and CO₂/H mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H₂-dependent conversion of CO₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for the production of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC) (see WO2009/094485). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or a downstream product related thereto such as an ester or amide thereof, including a nucleic acid encoding an aldehyde dehydrogenase of the invention, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the cells of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H₂ by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO₂ via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Acetyl-CoA can also be converted to acetoacetyl-CoA by, for example, acetoacetyl-CoA thiolase to funnel into a 1,3-BDO pathway, as disclosed herein (see FIG. 1). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway, or pathway to generate a downstream product related thereto such as an ester or amide thereof, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the cells of the invention can be performed such that the modified organism contains a reductive TCA pathway.

Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring cell can be produced that produces and/or secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, and any of the intermediate metabolites in the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the biosynthetic pathways for 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, including an aldehyde dehydrogenase of the invention. Accordingly, the invention provides a non-naturally occurring cell that produces and/or secretes 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway when grown on a carbohydrate or other carbon source. The cells producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, of the invention can initiate synthesis from an intermediate of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway.

The non-naturally occurring cells of the invention are constructed using methods well known in the art as exemplified herein to exogenously express an aldehyde dehydrogenase of the invention, and optionally at least one nucleic acid encoding a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway enzyme or protein, or a downstream product related thereto such as an ester or amide thereof. The enzymes or proteins can be expressed in sufficient amounts to produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. It is understood that the cells of the invention are cultured under conditions sufficient to express an aldehyde dehydrogenase of the invention or produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. Following the teachings and guidance provided herein, the non-naturally occurring cells of the invention can achieve biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, resulting in intracellular concentrations between about 0.1-300 mM or more, for example, 0.1-1.3 M or higher. Generally, the intracellular concentration of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring cells of the invention. For example, the intracellular concentration of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be between about 100 mM to 1.3 M, including about 100 mM, 200 mM, 500 mM, 800 mM, 1 M, 1.1 M, 1.2 M 1.3 M, or higher.

A cell of the invention is cultured using well known methods. The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these conditions can be employed with the non-naturally occurring cells as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO producers can synthesize 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO producing cells can produce 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, intracellularly and/or secrete the product into the culture medium.

As described herein, one exemplary growth condition for achieving biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring cells of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, an anaerobic condition refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, by a cell of the invention. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, will include culturing a non-naturally occurring cell producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the cell of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. Under anaerobic conditions, the culture is sparged with an inert gas or combination of gases, for example, nitrogen, N₂/CO₂ mixture, argon, helium, and the like. As the cells grow and utilize the carbon source, additional carbon source(s) and/or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and/or nutrients. The temperature of the bioreactor is maintained at a desired temperature, generally in the range of 22-37 degrees C., but the temperature can be maintained at a higher or lower temperature depending on the growth characteristics of the production organism and/or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3-6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the fermentation process.

In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and/or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and/or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and/or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired. Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art.

Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art and described herein.

In addition to the fermentation procedures described herein using the producers of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, of the invention for continuous production of substantial quantities of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide, producers also can be, for example, simultaneously subjected to chemical synthesis and/or enzymatic procedures to convert the product to other compounds, or the product can be separated from the fermentation culture and sequentially subjected to chemical and/or enzymatic conversion to convert the product to other compounds, if desired.

In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving expression of an aldehyde dehydrogenase of the invention or biosynthesis of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring cells of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a cell as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylsulfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a cell described herein from osmotic stress will depend on the cell used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM.

In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or any 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as “uptake sources.” Uptake sources can provide isotopic enrichment for any atom present in the product 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate, or for side products generated in reactions diverging away from a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13, and carbon-14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen-14 and nitrogen-15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased source derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon-14, or an environmental or atmospheric carbon source, such as CO₂, which can possess a larger amount of carbon-14 than its petroleum-derived counterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up for roughly 1 in 10¹² carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective Apr. 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio of carbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and M represent the ¹⁴C/¹²C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson, The use of Oxalic acid as a Standard. in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C² over C¹³ over C¹⁴, and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx H is −17.8 per mil. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0% represents the entire lack of carbon-14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm=100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a “modern” source includes biobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a “pre-bomb” standard, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50% water would be considered to have a Biobased Content=100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content=66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum-based product would be considered to have a Biobased Content=0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content of a compound or material and/or prepared downstream products that utilize a compound or material of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate, produced by a cell of the invention, that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is CO₂. In some embodiments, the present invention provides 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present invention provides 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically produced 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment. For example, in some aspects the invention provides bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a bioderived 3-HBal, 1,3-BDO, 4-HBal of 1,4-BDO intermediate having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or an intermediate of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products, which can be based on 3-HBal and/or 1,3-BDO, or a downstream product related thereto such as an ester or amide thereof, and plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, and the like, which can be based on 4-HBal and/or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about the same value as the CO₂ that occurs in the environment, wherein the plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene, and/or butadiene-based products are generated directly from or in combination with bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate as disclosed herein. Methods for producing butadiene and/or butadiene-based products have been described previously (see, for example, WO 2010/127319, WO 2013/036764, U.S. Pat. No. 9,017,983, US 2013/0066035, WO/2012/018624, US 2012/0021478, each of which is incorporated herein by reference). 1,3-BDO can be reacted with an acid, either in vivo or in vitro, to convert to an ester using, for example, a lipase. Such esters can have nutraceutical, pharmaceutical and food uses, and are advantaged when R-form of 1,3-BDO is used since that is the form (compared to S-form or the racemic mixture) best utilized by both animals and humans as an energy source (e.g., a ketone ester, such as (R)-3-hydroxybutyl-R-1,3-butanediol monoester (which has Generally Recognized As Safe (GRAS) approval in the United States) and (R)-3-hydroxybutyrate glycerol monoester or diester). The ketone esters can be delivered orally, and the ester releases R-1,3-butanediol that is used by the body (see, for example, WO2013150153). Methods of producing amides are well known in the art (see, for example, Goswami and Van Lanen, Mol. Biosyst. 11(2):338-353 (2015)).

Thus the present invention is particularly useful to provide an improved enzymatic route and microorganism to provide an improved composition of 1,3-BDO, namely R-1,3-butanediol, highly enriched or essentially enantiomerically pure, and further having improved purity qualities with respect to by-products. 1,3-BDO has further food related uses including use directly as a food source, a food ingredient, a flavoring agent, a solvent or solubilizer for flavoring agents, a stabilizer, an emulsifier, and an anti-microbial agent and preservative. 1,3-BDO is used in the pharmaceutical industry as a parenteral drug solvent. 1,3-BDO finds use in cosmetics as an ingredient that is an emollient, a humectant, that prevents crystallization of insoluble ingredients, a solubilizer for less-water-soluble ingredients such as fragrances, and as an anti-microbial agent and preservative. For example, it can be used as a humectant, especially in hair sprays and setting lotions; it reduces loss of aromas from essential oils, preserves against spoilage by microorganisms, and is used as a solvent for benzoates. 1,3-BDO can be used at concentrations from 0.1% to 50%, and even less than 0.1% and even more than 50%. It is used in hair and bath products, eye and facial makeup, fragrances, personal cleanliness products, and shaving and skin care preparations (see, for example, the Cosmetic Ingredient Review board's report: “Final Report on the Safety Assessment of Butylene Glycol, Hexylene Glycol, Ethoxydiglycol, and Dipropylene Glycol”, Journal of the American College of Toxicology, Volume 4, Number 5, 1985, which is incorporated herein by reference). This report provides specific uses and concentrations of 1,3-BDO in cosmetics; see for examples the report's Table 2 therein entitled “Product Formulation Data”.

In one embodiment, the invention provides culture medium comprising bioderived 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO, wherein the bioderived 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO, has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, and wherein the bioderived 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO is produced by a cell, or in a cell lysate, of the invention or a method of the invention. In one embodiment, the culture medium is separated from the cell.

In one embodiment, the invention provides 3-hydroxybutyraldeyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or 4-hydroxybutyraldeyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, wherein the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, is produced by a cell, or in a cell lysate, of the invention or a method of the invention. In one embodiment, the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

In one embodiment, the invention provides 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), produced by a cell, or in a cell lysate of the invention or a method of the invention. In one embodiment, the invention provides 3-hydroxybutyraldeyde (3-HBal) and/or 1,3-butanediol (1,3-BDO) having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, wherein the 3-HBal and/or 1,3-BDO is produced by a cell, or in a cell lysate, of the invention or a method of the invention, wherein the 3-HBal and/or 1,3-BDO is enantiomerically enriched for the R form. In one embodiment, the 3-HBal and/or 1,3-BDO has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

In one embodiment, the invention provides 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO) produced by a cell, or in a cell lysate, of the invention or a method of the invention, wherein the 3-HBal and/or 1,3-BDO is enantiomerically enriched for the R form. In one embodiment, the R form is greater than 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of the 3-HBal and/or 1,3-BDO. In one embodiment, the 3-HBal and/or 1,3-BDO is ≥55% R-enantiomer, —≥60% R-enantiomer, ≥65% R-enantiomer, ≥70% R-enantiomer, ≥75% R-enantiomer, ≥80% R-enantiomer, ≥85% R-enantiomer, ≥90% R-enantiomer, or ≥95% R-enantiomer, and can be highly chemically pure, e.g., ≥99%, for example, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8% or ≥99.9% R-enantiomer.

In one embodiment, the invention provides a composition comprising 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, produced by a cell, or in a cell lysate, of the invention or a method of the invention and a compound other than the 3-HBal and/or 1,3-BDO, or 4-HBal or 1,4-BDO, respectively. In one embodiment, the compound other than the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, is a portion of a cell that produces the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, respectively, or that expresses a polypeptide of the invention.

In one embodiment, the invention provides a composition comprising 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, produced by a cell, or in a cell lysate, of the invention or a method of the invention, or a cell lysate or culture supernatant of a cell producing the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO.

In one embodiment, the invention provides a product comprising 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, produced by a cell, or in a cell lysate of the invention or a method of the invention, wherein the product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-hydroxybutyrate (P4HB) or a co-polymer thereof, poly(tetramethylene ether) glycol (PTMEG), polybutylene terephthalate (PBT), polyurethane-polyurea copolymer, nylon, organic solvent, polyurethane resin, polyester resin, hypoglycaemic agent, butadiene or butadiene-based product. In one embodiment, the product is a cosmetic product or a food additive. In one embodiment, the product comprises at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived 3-HBal and/or 1,3-BDO, or bioderived 4-HBal and/or 1,4-BDO. In one embodiment, the product comprises a portion of the produced 3-HBal and/or 1,3-BDO, or the produced 4-HBal and/or 1,4-BDO, as a repeating unit. In one embodiment, the invention provides a molded product obtained by molding a product made with or derived from 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO produced by a cell, or in a cell lysate of the invention or a method of the invention.

The invention further provides a composition comprising bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, and a compound other than the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. The compound other than the bioderived product can be a cellular portion, for example, a trace amount of a cellular portion of, or can be fermentation broth or culture medium or a purified or partially purified fraction thereof produced in the presence of, a non-naturally occurring cell of the invention having a pathway that produces 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. The composition can comprise, for example, a reduced level of a byproduct when produced by an organism having reduced byproduct formation, as disclosed herein. The composition can comprise, for example, bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or a cell lysate or culture supernatant of a cell of the invention.

3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, is a chemical used in commercial and industrial applications. Non-limiting examples of such applications include production of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products. Moreover, 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO is also used as a raw material in the production of a wide range of products including plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products. Accordingly, in some embodiments, the invention provides biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products comprising one or more bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate produced by a non-naturally occurring cell of the invention, for example, expressing an aldehyde dehydrogenase of the invention, or produced using a method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the cells of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term “biobased” means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products comprising bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate, wherein the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate includes all or part of the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate used in the production of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products. For example, the final plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products can contain the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate, or a portion thereof that is the result of the manufacturing of plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products. Such manufacturing can include chemically reacting the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate (e.g. chemical conversion, chemical functionalization, chemical coupling, oxidation, reduction, polymerization, copolymerization and the like) into the final plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products. Thus, in some aspects, the invention provides a biobased plastic, elastic fiber, polyurethane, polyester, including polyhydroxyalkanoate such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymer, referred to as spandex, elastane or Lycra™, nylon, polyurethane resin, polyester resin, hypoglycaemic agent, butadiene and/or butadiene-based product comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate as disclosed herein.

Additionally, in some embodiments, the invention provides a composition having a bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate disclosed herein and a compound other than the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate. For example, in some aspects, the invention provides biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products wherein the 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate used in its production is a combination of bioderived and petroleum derived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate. For example, biobased plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products can be produced using 50% bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, and 50% petroleum derived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the cells disclosed herein. It is understood that methods for producing plastics, elastic fibers, polyurethanes, polyesters, including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO, polytetramethylene oxide), polybutylene terephthalate (PBT), and polyurethane-polyurea copolymers, referred to as spandex, elastane or Lycra™, nylons, organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene-based products using the bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, or bioderived 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO pathway intermediate of the invention are well known in the art.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof.

One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring cells for further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication 2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed Jun. 14, 2002, and in International Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host cells. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

As disclosed herein, the invention relates to aldehyde dehydrogenase variants (see Example). The generation of such variants is described in the Example. Any of a variety of methods can be used to generate an aldehyde dehydrogenase variant such as the aldehyde dehydrogenase variants disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described below (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014).

As disclosed herein, a nucleic acid encoding a desired activity of a pathway for 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof, pathway enzyme or protein to increase production of 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product related thereto such as an ester or amide thereof. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >10⁴). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., Appl. Biochem. Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K_(m)), including broadening substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a pathway enzyme or protein for producing 3-HBal, 1,3-BDO, 4-HBal or 1,4-BDO, or a downstream product thereof such as an ester or amide thereof, or an aldehyde dehydrogenase of the invention. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Theor. Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751(1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad Sci. USA 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al., Biotechnol. J 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode “all genetic diversity in targets” and allows a very high diversity for the shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al., Angew. Chem. Int. EdEngl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-× in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al., Appl. Environ Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad Sci. USA 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad Sci. USA 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego Calif.), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. EdEngl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

Example Aldehyde Dehydrogenase Variants

This example describes generation of aldehyde dehydrogenase variants with desirable properties.

Mutagenesis techniques were used to generate variant aldehyde dehydrogenases based on template ALD-1. Variants were generated using error prone PCR, site directed mutagenesis, and by spontaneous mutations during genetic selection. Template ALD-1 corresponds to the aldehyde dehydrogenase provided below:

(SEQ ID NO: 1) MIKDTLVSITKDLKLKTNVENANLKNYKDDSSCFGVFENVENAISNAVH AQKILSLHYTKEQREKIITEIRKAALENKEILATMILEETHMGRYEDKI LKHELVAKYTPGTEDLTTTAWSGDNGLTVVEMSPYGVIGAITPSTNPIE TVICNSIGMTAAGNTVVFNGHPGAKKCVAFAVEMINKAIISCGGPENLV TTIKNPTMDSLDAIIKHPSIKLLCGTGGPGMVKTLLNSGKKAIGAGAGN PPVIVDDTADIEKAGKSIIEGCSFDNNLPCIAEKEVFVFENVADDLISN MLKNNAVIINEDQVSKLIDLVLQKNNETQEYSINKKWVGKDAKLFLDEI DVESPSSVKCIICEVSASHPFVMTELMMPILPIVRVKDIDEAIEYAKIA EQNRKHSAYIYSKNIDNLNRFEREIDTTIFVKNAKSFAGVGYEAEGFTT FTIAGSTGEGITSARNFTRQRRCVLAG.

Additional ALD sequences for ALD-2 and ALD-3 are provided below:

ALD-2 (SEQ ID NO: 2) MNTENIEQAIRKILSEELSNPQSSTATNTTVPGKNGIFKTVNEATAATK AAQENYADQPISVRNKVIDAIREGFRPYIEDMAKRIHDETGMGTVSAKI AKLNNALYNTPGPEILQPEAETGDGGLVMYEYAPFGVIGAVGPSTNPSE TVIANAIMMLAGGNTLFFGAHPGAKNITRWTIEKLNELVADATGLHNLV VSLETPSIESVQEVMQHPDVAMLSITGGPAVVHQALISGKKAVGAGAGN PPAMVDATANIALAAHNIVDSAAFDNNILCTAEKEVVVEAAVKDELIMR MQQEGAFLVTDSADIEKLAQMTIGPKGAPDRKFVGKDATYILDQAGISY TGTPTLIILEAAKDHPLVTTEMLMPILPVVCCPDFDSVLATATEVEGGL HHTASIHSENLPHINKAAHRLNTSIFVVNGPTYCGTGVATNGAHSGASA LTIATPTGEGTATSKTYTRRRRLNSPEGFSLRTWEA ALD-3 (SEQ ID NO: 3) MTVNEQLVQDIIKNVVASMQLTQTNKTELGVFDDMNQAIEAAKEAQLVV KKMSMDQREKIISAIRKKTIEHAETLARMAVEETGMGNVGHKILKHQLV AEKTPGTEDITTTAWSGDRGLTLVEMGPFGVIGAITPCTNPSETIICNT IGMLAGGNTVVFNPHPAAIKTSNFAVQLINEASLSAGGPVNIACSVRKP TLDSSKIMMSHQDIPLIAATGGPGVVTAVLQSGKRGIGAGAGNPPVLVD ETADIRKAAEDIINGCTFDNNLPCIAEKEVVAIDAIANELMNYMVKEQG CYAITKEQQEKLTNLVITPKGLNRNCVGKDARTLLGMIGIDVPSNIRCI IFEGEKEHPLISEELMMPILGIVRAKSFDDAVEKAVWLEHGNRHSAHIH SKNVDRITTYAKAIDTAILVKNAPSYAAIGFGGEGFCTFTIASRTGEGL TSASTFTKRRRCVMSDSLCIR

ALD-1 is slightly more specific for the R enantiomer of 3-hydroxybutyryl-CoA compared to the S enantiomer. A sequence alignment of ALD-1 to ALD-2 and ALD-3 is shown in FIG. 3. The sequences correspond to SEQ ID NOS:1, 2 and 3, respectively. A crystal structure also exists for ALD-3 (PDBID 4C3S), and ALD-2 is more closely related to ALD-3 than ALD-1. Therefore ALD-3 was used as the template. Underlined in FIG. 3 are 2 loop regions, the first designated A, the second B, both involved in substrate specificity and enantiomer specificity as determined herein. Loop A in ALD-1 is sequence LQKNNETQEYSINKKWVGKD (SEQ ID NO:124), in ALD-2 is sequence IGPKGAPDRKFVGKD (SEQ ID NO:125) and in ALD-3 is sequence ITPKGLNRNCVGKD (SEQ ID NO:126). Loop B in ALD-1 is sequence SFAGVGYEAEGFTTFTIA (SEQ ID NO:127), in ALD-2 is sequence TYCGTGVATNGAHSGASALTIA (SEQ ID NO:128), and in ALD-3 is sequence SYAAIGFGGEGFCTFTIA (SEQ ID NO:129). The sequence and the length of the substrate specificity loop A and B from ALD-2 differs from those of ALD-1 and ALD-3; nevertheless the alignment shows sufficient conservation to facilitate identification of corresponding positions for substitution as described herein, and especially so if combined with 3D modeling as shown in FIG. 6, which shows the two loop regions interacting to affect substrate specificity and enantiomer specificity, especially when modified with exemplary substitutions as described herein. ALD-1 and ALD-3 are 51.9% identical. ALD-1 and ALD-2 are 35.9% identical. ALD-3 and ALD-2 are 40% identical. A consensus ALD sequence based on the alignment of FIG. 3 was generated. A consensus for Loop A based on alignment of ALD-1, ALD-2 and ALD-3 is IXPKG----XXNRKXVGKD (SEQ ID NO:5). A consensus for Loop B based on alignment of ALD-1, ALD-2 and ALD-3 is SYAGXGXXXE----GFXTFTIA (SEQ ID NO:6).

Additional alignments were performed (FIG. 4). FIG. 4A shows an alignment with a 40-55% cutoff compared to ALD-1. FIG. 4B shows an alignment with a 75-90% cutoff compared to ALD-1. FIG. 4C shows an alignment with a 90% cutoff compared to ALD-1. The alignments of exemplary aldehyde deydrogenases (ALD) shown in FIGS. 4A-4C demonstrate identifying positions in ALDs that correspond to positions in the representative template ALD sequence where substitutions of the invention can be made. Underlined are two key loop regions, the first designated A, the second B, both involved in substrate specificity and enantiomer specificity as determined herein. FIGS. 4A-4C demonstrate that corresponding positions for substitutions taught herein can be identified in ALDs that are at least 40% identical with ALD-1, especially the Loop A and B regions, and especially the very conserved Loop B region.

Mutagenesis to increase the specificity of variant 45 for 3HB-CoA relative to acetyl-CoA led to several variants with increased 1,3 BDO production and decreased ethanol. Mutations that increase specificity of 3-hydroxybutyryl-CoA over acetyl-CoA provide a decrease in ethanol, since the acetaldehyde generated from acetyl-CoA can be converted to ethanol by enzymes natively in the host cell or by a pathway enzyme that converts 3-hydroxybutyraldehyde to 1,3-butanediol. Variants that increase enzymatic activity of aldehyde dehydrogenase or increase its specificity for 3-hydroxybutyryl-CoA decrease 4-hydroxy-2-butanone by increasing flux through an enzymatic pathway to 1,3-butanediol which pulls acetoacetyl-CoA towards 1,3-butanediol formation, decreasing its availability for two-step conversion to 4-hydroxy-2-butanone by native enzymes or less-specific pathway enzymes. The sequence of variant 45 is provided below:

(SEQ ID NO: 4) MIKDTLVSITKDLKLKTNVENANLKNYKDDSSCFGVFENVENAISNAVH AQKILSLHYTKEQREKIITEIRKAALENKEILATMILEETHMGRYEDKI LKHELVAKYTPGTEDLTTTAWSGDNGLTVVEMSPYGVIGAITPSTNPIE TVICNSIGMTAAGNTVVFNGHPGAKKSVAFAVEMINKAIISCGGPENLV TTIKNPTRDSLDAIIKHPSIKLLVGTGGPGMVKTLLNSGKKAIGAGAGN PPVIVDDTADIEKAGKSIIEGASFDNNLPCIAEKEVFVFENVADDLISN MLKNNAVIINEDQVSKLIDLVLQKNNETQEYSINKKWVGKDAKLFLDEI DVESPSSVKCIITEVSASHPFVMTELMMPILPIVRVKDIDEAIEYAKIA EQNHKHSAYIYSKNIDNLNRFEREIDTTIFVKNAKSFAGVGYEAPGFTT FTIAGSTGEGITSARNFTRQRRIVLVG

The assay performed is an in vitro assay to examine the activity on 3HB-CoA by monitoring a decrease in absorbance as NADH is converted to NAD. Assays were also performed with acetyl-CoA (AcCoA) as a substrate, and improved enzymes were identified as an improvement in the ratio of activity for 3HB-CoA vs. AcCoA. Mutations that increase specificity of 3-hydroxybutyryl-CoA over acetyl-CoA provide a decrease in ethanol, since the acetaldehyde generated from acetyl-CoA can be converted to ethanol by enzymes natively in the host cell or by a pathway enzyme that converts 3-hydroxybutyraldehyde to 1,3-butanediol.

Further investigation of a subset of these variants with (R) and (S) 3-hydroxybutyraldehyde showed that five of the tested variants (952, 955, 957, 959, 961) had improved selectivity for the R enantiomer compared to the parent enzyme (variant 45) and wildtype ALD-1 (FIG. 5). FIG. 5A shows the specific activity of ALD-2, ALD-1 and ALD-1 variants on 3 hydroxy-(R)-butyraldehyde (left bars in sets of bars) and 3 hydroxy-(S)-butyraldehyde (right bars in sets of bars). Purified streptavidin-tagged proteins were assayed at 35° C. in IVI buffer pH 7.5, 0.5 mM NAD⁺, 2 mM CoA in the presence of either 10 mM R or S 3-hydroxybutyraldehyde, and activity was monitored by change in NADH absorbance at 340 nm. IV buffer contains 5 mM potassium phosphate monobasic, 20 mM potassium phosphate dibasic, 10 mM sodium glutamate, monohydrate, and 150 mM potassium chloride, pH 7.5. Thus, the enzyme reaction in the assay was carried out in the reverse direction from that shown in FIG. 1, that is, the reaction measured the conversion of 3-hydroxybutyraldehyde to 3-hydroxybutyryl-CoA. As shown in FIG. 5B, certain aldehyde dehydrogenase variants exhibited selectivity for R-3-hydroxybutyraldehyde (R-3HB-aldehyde) over S-3-hydroxybutyraldehyde (S-3HB-aldehyde).

Computational modeling of the mutant 959 using an ALD-1 crystal structure suggests that the amino acid substitution F442N allows a hydrogen bond network to be formed with the hydroxyl on carbon 3 of the R isomer but not the (S) isomer (FIG. 6). FIGS. 6A-6C show ribbon diagrams of the structure of the aldehyde dehydrogenase 959. The diagrams show docking of 3-hydroxy-(R)-butyraldehyde (FIG. 6A) or 3-hydroxy-(S)-butyraldehyde (FIG. 6B) into the structure of 959. FIG. 6C shows that when the 3-hydroxy-(S)-butyraldehyde is docked in the same orientation most energetically favored for docking of 3-hydroxy-(R)-butyraldehyde as shown in FIG. 6A an unfavorable interaction (circled) is created with an isoleucine located in the active site. The model indicates that mutation F442N creates a hydrogen bond between the protein and a hydroxyl of 3-hydroxy-(R)-butyraldehyde that is not possible with the S enantiomer.

Exemplary aldehyde dehydrogenase variants are shown in Tables 1A-1D.

TABLE 1A Exemplary ALD Variants Position Variant 12 19 33 44 65 66 72 73 107 122 129 139 143 12 D12A I139S 16 D12A C33R I139S 17 D12A I139V T143N 30 E129I 34 D12A I139S 56 D12A I139S 71 Y107K 80 Y107K 93 D12A I139S 156 D12A Y107K 166 D12A Y107K 180 D12A I139S 182 184 D12A I139S 194 I139S 199 203 205 D12A I139S 208 213 T143S 235 D12A I139S 240 D12A I139V 321 D12V I139S 331 K65A I66M 598 D12A I139S 601 K65A I66Q 602 K65A I66N 603 K65A I66H 604 K65A I66T 605 K65A I66S 45 681 K65A I66M A73S 682 K65A I66Q A73S 683 K65A I66N A73S 684 K65A I66H A73S 685 K65A I66T A73S 686 K65A I66S A73S 687 688 K65A 721 I66M 722 I66Q 723 I66N 724 I66H 725 I66T 726 I66S 775 I66Q 776 I66N 777 I66H 778 I66T 779 I66S 780 I66M 781 K65A 782 K65A I66M 783 D12A I66M I139V 784 D12A K65A I139V 785 D12A K65A I66M I139V 921 K65A I66Q 922 K65A I66N 923 K65A I66H 924 K65A I66T 925 K65A I66S 951 952 953 954 955 956 957 958 959 960 V19I D122N 961 975 D12A I139V 991 D12A I139L T143N 992 A73S 993 994 995 996 997 I44L 998 999 K65A 1000 1001 1002 1003 1004 1005 1006 1007 I66M 1008 K65A 1009 K65A I66M 1011 1012 I66M 1013 K65A 1014 K65A I66M 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 K72N 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 A73D 1080 A73G 1081 A73L 1082 A73Q 1083 A73F 1084 A73G 1085 A73E 1086 A73W 1087 1088 1089 1090 1091 1092 1093 A73L 1094 A73R 1095 A73C 1096 1097 A73W 1098 A73M 1099 1100 A73F 1101

TABLE 1B Exemplary ALD Variants Position Variant 145 155 163 167 174 189 204 220 227 229 12 M204R 16 C174S C189A M204R C220V 17 G167S C174S M204R C220V 30 C174S C220V 34 C174S M204R C220V 56 C174S M204R C220V 71 C174S M204R C220V 80 C174S C220V 93 C174S M204R C220V 156 C174S M204R C220V 166 C174S C220V 180 C174S M204R C220V 182 C174S M204R C220V 184 C174S M204R C220V 194 C174S M204R C220V 199 C174S M204R C220V 203 C174S M204R C220V 205 C174S M204R C220V 208 C174S M204R C220V 213 C174S M204R C220V 235 C174S M204R C220V 240 C174S M204R C220V M227K 321 M204R 331 C174S M204R C220V 598 C174S M204R C220V M227Q 601 C174S M204R C220V 602 C174S M204R C220V 603 C174S M204R C220V 604 C174S M204R C220V 605 C174S M204R C220V 45 C174S M204R C220V 681 C174S M204R C220V M227I 682 C174S M204R C220V M227I 683 C174S M204R C220V M227I 684 C174S M204R C220V M227I 685 C174S M204R C220V M227I 686 C174S M204R C220V M227I 687 C174S M204R C220V 688 C174S M204R C220V 721 C174S M204R C220V 722 C174S M204R C220V 723 C174S M204R C220V 724 C174S M204R C220V 725 C174S M204R C220V 726 C174S M204R C220V 775 C174S M204R C220V 776 C174S M204R C220V 777 C174S M204R C220V 778 C174S M204R C220V 779 C174S M204R C220V 780 C174S M204R C220V 781 C174S M204R C220V 782 C174S M204R C220V 783 C174S M204R C220V M227Q 784 C174S M204R C220V M227Q 785 C174S M204R C220V M227Q 921 C174S M204R C220V 922 C174S M204R C220V 923 C174S M204R C220V 924 C174S M204R C220V 925 C174S M204R C220V 951 C174S M204R C220V 952 C174S M204R C220V 953 C174S M204R C220V 954 C174S M204R C220V 955 C174S M204R C220V 956 C174S M204R C220V 957 C174S M204R C220V 958 C174S M204R C220V 959 C174S M204R C220V 960 C174S M204R C220V 961 C174S M204R C220V 975 C174S M204R C220V M227Q 991 C174S M204R C220V 992 C174S M204R C220V 993 C174S M204R C220V 994 V163C C174S M204R C220V 995 C174S M204R C220V K 229S 996 C174S M204R C220V 997 C174S M204R C220V 998 C174S M204R C220V 999 C174S M204R C220V 1000 V163C C174S M204R C220V 1001 C174S M204R C220V 1002 C174S M204R C220V 1003 G155G C174S M204R C220V 1004 P145P C174S M204R C220V 1005 C174S M204R C220V 1006 C174S M204R C220V 1007 C174S M204R C220V 1008 C174S M204R C220V 1009 C174S M204R C220V 1011 C174S M204R C220V 1012 C174S M204R C220V 1013 C174S M204R C220V 1014 C174S M204R C220V 1015 C174S M204R C220V M227I 1016 C174S M204R C220V 1017 C174S M204R C220V 1018 C174S M204R C220V 1019 C174S M204R C220V 1020 C174S M204R C220V 1021 C174S M204R C220V M227V 1022 C174S M204R C220V M227V 1023 C174S M204R C220V M227I 1024 C174S M204R C220V M227I 1025 C174S M204R C220V 1026 C174S M204R C220V 1027 C174S M204R C220V M227I 1028 C174S M204R C220V 1029 C174S M204R C220V 1030 C174S M204R C220V 1031 C174S M204R C220V 1032 C174S M204R C220V 1033 C174S M204R C220V 1034 C174S M204R C220V M227I 1035 C174S M204R C220V 1036 C174S M204R C220V 1037 C174S M204R C220V 1038 C174S M204R C220V 1039 C174S M204R C220V 1040 C174S M204R C220V 1041 C174S M204R C220V 1042 C174S M204R C220V 1043 C174S M204R C220V M227V 1044 C174S M204R C220V 1045 C174S M204R C220V 1046 C174S M204R C220V 1047 C174S M204R C220V M227C 1048 C174S M204R C220V M227L 1049 C174S M204R C220V 1050 C174S M204R C220V M227C 1051 C174S M204R C220V 1052 C174S M204R C220V 1053 C174S M204R C220V M227C 1054 C174S M204R C220V M227C 1055 C174S M204R C220V 1056 C174S M204R C220V 1057 C174S M204R C220V 1058 C174S M204R C220V 1059 C174S M204R C220V 1060 C174S M204R C220V M227L 1061 C174S M204R C220V M227A 1062 C174S M204R C220V 1063 C174S M204R C220V 1064 C174S M204R C220V 1065 C174S M204R C220V 1066 C174S M204R C220V M227I 1067 C174S M204R C220V M227I 1068 C174S M204R C220V M227I 1069 C174S M204R C220V 1070 C174S M204R C220V M227V 1071 C174S M204R C220V M227C 1072 C174S M204R C220V 1073 C174S M204R C220V 1074 C174S M204R C220V 1075 C174S M204R C220V 1076 C174S M204R C220V M227L 1077 C174S M204R C220V 1078 C174S M204R C220V M227V 1079 C174S M204R C220V M227I 1080 C174S M204R C220V M227I 1081 C174S M204R C220V M227I 1082 C174S M204R C220V M227I 1083 C174S M204R C220V M227I 1084 C174S M204R C220V M227I 1085 C174S M204R C220V M227I 1086 C174S M204R C220V M227I 1087 V163G C174S M204R C220V M227I 1088 V163T C174S M204R C220V M227I 1089 C174S M204R C220V M227L 1090 C174S M204R C220V 1091 C174S M204R C220V 1092 C174S M204R C220V 1093 C174S M204R C220V M227I 1094 C174S M204R C220V M227I 1095 V163C C174S M204R C220V M227I 1096 V163C C174S M204R C220V M227I 1097 V163C C174S M204R C220V M227I 1098 V163C C174S M204R C220V M227I 1099 V163C C174S M204R C220V M227I 1100 V163C C174S M204R C220V M227I 1101 V163C C174S M204R C220V M227I

TABLE 1C Exemplary ALD Variants Position Variant 230 243 244 254 267 315 353 356 396 429 12 R396H 16 C267A C353A C356T R396H 17 T230R C267A C356T R396H F429Y 30 C267A C356T R396H 34 C267A C356T R396H 56 C267A C356T R396H F429Y 71 C267A C356T 80 C267A C356T 93 T230R C267A C356T R396H F429Y 156 C267A C356T 166 C267A C356T 180 C267A C356T R396H 182 A243P C267A C356T R396H 184 C267A C356T R396H 194 C267A C356T R396H 199 C267A C356T R396H F429Q 203 C267A C356T R396H F429Y 205 A243P C267A C356T R396H F429Y 208 C267A C356T R396H 213 C267A C356T R396H 235 A243P C267A C356T R396H 240 C267A C356T R396H F429Y 321 R396H 331 A243Q C267A C356T R396H 598 T230R A243P C267A C356T R396H F429Y 601 A243Q C267A C356T R396H 602 A243Q C267A C356T R396H 603 A243Q C267A C356T R396H 604 A243Q C267A C356T R396H 605 A243Q C267A C356T R396H 45 C267A C356T R396H 681 T230C A243P C267A C356T R396H 682 T230C A243P C267A C356T R396H 683 T230C A243P C267A C356T R396H 684 T230C A243P C267A C356T R396H 685 T230C A243P C267A C356T R396H 686 T230C A243P C267A C356T R396H 687 C267A C356T R396H 688 A243Q C267A C356T R396H 721 A243Q C267A C356T R396H 722 A243Q C267A C356T R396H 723 A243Q C267A C356T R396H 724 A243Q C267A C356T R396H 725 A243Q C267A C356T R396H 726 A243Q C267A C356T R396H 775 A243P C267A C356T R396H 776 A243P C267A C356T R396H 777 A243P C267A C356T R396H 778 A243P C267A C356T R396H 779 A243P C267A C356T R396H 780 C267A C356T R396H F429H 781 C267A C356T R396H F429H 782 C267A C356T R396H F429H 783 T230R A243P C267A C356T R396H F429Y 784 T230R A243P C267A C356T R396H F429Y 785 T230R A243P C267A C356T R396H F429Y 921 A243P C267A C356T R396H 922 A243P C267A C356T R396H 923 A243P C267A C356T R396H 924 A243P C267A C356T R396H 925 A243P C267A C356T R396H 951 C267A C356T R396H F429H 952 C267A C356T R396H F429M 953 C267A C356T R396H F429M 954 C267A C356T R396H F429Q 955 C267A C356T R396H 956 C267A C356T R396H 957 C267A C356T R396H 958 C267A C356T R396H 959 C267A C356T R396H 960 C267A C356T R396H F429D 961 C267A V315A C356T R396H 975 T230R A243P C267A C356T R396H F429Y 991 T230R A243P C267A C356T R396H F429Y 992 C267A C356T R396H 993 A254T C267A C356T R396H 994 C267A C356T R396H 995 C267A C356T R396H 996 C267A C356L R396H 997 C267A C356T R396H 998 C267A C356T R396H 999 C267A C356T R396H 1000 C267A C356T R396H 1001 C267A C356T R396H 1002 C267A C356T R396H 1003 C267A C356T R396H 1004 C267A C356T R396H 1005 G244G C267A C356T R396H 1006 C267A C356T R396H 1007 C267A C356T R396H 1008 C267A C356T R396H 1009 C267A C356T R396H 1011 A243P C267A C356T R396H 1012 A243P C267A C356T R396H 1013 A243P C267A C356T R396H 1014 A243P C267A C356T R396H 1015 T230K C267A C356T R396H 1016 T230R A243Q C267A C356T R396H 1017 T230H A243Q C267A C356T R396H 1018 T230A A243E C267A C356T R396H 1019 T230M A243S C267A C356T R396H 1020 T230H A243N C267A C356T R396H 1021 T230C C267A C356T R396H 1022 T230H C267A C356T R396H 1023 T230L C267A C356T R396H 1024 T230C C267A C356T R396H 1025 T230M A243E C267A C356T R396H 1026 T230S A243Q C267A C356T R396H 1027 T230A C267A C356T R396H 1028 T230K C267A C356T R396H 1029 T230Y A243Q C267A C356T R396H 1030 T230G A243Q C267A C356T R396H 1031 T230M A243K C267A C356T R396H 1032 T230T A243L C267A C356T R396H 1033 T230I C267A C356T R396H 1034 T230K C267A C356T R396H F429L 1035 T230H C267A C356T R396H 1036 T230Y A243E C267A C356T R396H 1037 A243S C267A C356T R396H 1038 T230C A243K C267A C356T R396H 1039 T230H A243K C267A C356T R396H 1040 T230H A243C C267A C356T R396H 1041 T230A A243Q C267A C356T R396H 1042 T230S A243C C267A C356T R396H 1043 T230S C267A C356T R396H 1044 T230H A243M C267A C356T R396H 1045 T230A A243K C267A C356T R396H 1046 T230W C267A C356T R396H 1047 T230R C267A C356T R396H 1048 T230N C267A C356T R396H 1049 T230N C267A C356T R396H 1050 T230L C267A C356T R396H 1051 T230V C267A C356T R396H 1052 T230L C267A C356T R396H 1053 T230K C267A C356T R396H 1054 T230V C267A C356T R396H 1055 T230T A243N C267A C356T R396H 1056 T230T A243I C267A C356T R396H 1057 T230T A243C C267A C356T R396H 1058 T230G A243K C267A C356T R396H 1059 T230R A243K C267A C356T R396H 1060 A243P C267A C356T R396H 1061 A243P C267A C356T R396H 1062 A243Q C267A C356T R396H 1063 T230Q C267A C356T R396H 1064 T230N A243I C267A C356T R396H 1065 T230C A243C C267A C356T R396H 1066 T230R C267A C356T R396H 1067 A243L C267A C356T R396H 1068 A243M C267A C356T R396H 1069 A243M C267A C356T R396H 1070 C267A C356T R396H 1071 A243Q C267A C356T R396H 1072 T230R A243C C267A C356T R396H 1073 T230L A243M C267A C356T R396H 1074 T230I A243M C267A C356T R396H 1075 T230M A243Q C267A C356T R396H 1076 T230W C267A C356T R396H 1077 T230V A243M C267A C356T R396H 1078 T230I C267A C356T R396H 1079 T230K C267A C356T R396H 1080 T230K C267A C356T R396H 1081 T230K C267A C356T R396H 1082 T230K C267A C356T R396H 1083 T230K C267A C356T R396H 1084 T230K C267A C356T R396H 1085 T230K C267A C356T R396H 1086 T230K C267A C356T R396H 1087 T230K C267A C356T R396H 1088 T230K C267A C356T R396H 1089 T230S C267A C356T R396H 1090 A243E C267A C356T R396H 1091 T230T A243E C267A C356T R396H 1092 A243K C267A C356T R396H 1093 T230K C267A C356T R396H 1094 T230K C267A C356T R396H 1095 T230K C267A C356T R396H 1096 T230K C267A C356T R396H 1097 T230K C267A C356T R396H 1098 T230K C267A C356T R396H 1099 T230K C267A C356T R396H 1100 T230K C267A C356T R396H 1101 T230K C267A C356T R396H

TABLE 1D Exemplary ALD Variants Position Variant 432 437 440 441 442 444 447 450 460 464 467 12 16 C464V 17 E437P F442T C464I A467V 30 C464I A467V 34 C464I 56 E437P F442T C464I A467V 71 C464I A467V 80 C464I 93 E437P F442T C464I A467V 156 C464I A467V 166 C464I 180 C464I A467V 182 E437P C464I A467V 184 E437P C464I A467V 194 E437P C464I A467V 199 E437P C464I A467V 203 E437P F442T C464I A467V 205 E437P F442T C464I A467V 208 E437P F442Y C464I A467V 213 E437P C464I A467V 235 E437P C464I A467V 240 E437P F442T C464I A467V 321 331 E437P F442N C464I A467V 598 E437P F442T C464I A467V 601 E437P F442N C464I A467V 602 E437P F442N C464I A467V 603 E437P F442N C464I A467V 604 E437P F442N C464I A467V 605 E437P F442N C464I A467V 45 E437P C464I A467V 681 E437P F442N S447P C464I A467V 682 E437P F442N S447P C464I A467V 683 E437P F442N S447P C464I A467V 684 E437P F442N S447P C464I A467V 685 E437P F442N S447P C464I A467V 686 E437P F442N S447P C464I A467V 687 E437P F442M C464I A467V 688 E437P F442N C464I A467V 721 E437P F442N C464I A467V 722 E437P F442N C464I A467V 723 E437P F442N C464I A467V 724 E437P F442N C464I A467V 725 E437P F442N C464I A467V 726 E437P F442N C464I A467V 775 E437P F442N C464I A467V 776 E437P F442N C464I A467V 777 E437P F442N C464I A467V 778 E437P F442N C464I A467V 779 E437P F442N C464I A467V 780 E437P F442H C464I A467V 781 E437P F442H C464I A467V 782 E437P F442H C464I A467V 783 E437P F442T C464I A467V 784 E437P F442T C464I A467V 785 E437P F442T C464I A467V 921 E437P F442N C464I A467V 922 E437P F442N C464I A467V 923 E437P F442N C464I A467V 924 E437P F442N C464I A467V 925 E437P F442N C464I A467V 951 E437P F442H C464I A467V 952 E437P F442H C464I A467V 953 E437P F442N C464I A467V 954 E437P C464I A467V 955 E437P F442N C464I A467V 956 E437P F442N C464I A467V 957 E437P F442Q C464I A467V 958 E437P i444V C464I A467V 959 E437P T440H F442N C464I A467V 960 E437P F442Q E450E C464I A467V 961 E437P T440H F442N C464I A467V 975 E437P F442T C464I A467V 991 E437P F442T C464I A467V 992 E437P F442M S447M C464I A467V 993 E437P F442M C464I A467V 994 E437P F442M C464I A467V 995 E437P F442N C464I A467V 996 E437P F442N C464I A467V 997 E437P T441G C464I A467V 998 E437P F442M C464I A467V 999 E437P F442N C464I A467V 1000 E437P F442N C464I A467V 1001 E437P F442M R460K C464I A467V 1002 E437P F442M S447M C464I A467V 1003 E437P F442F C464I A467V 1004 E437P C464I A467V 1005 E437P C464I A467V 1006 V432V E437P C464I A467V 1007 V432V E437P C464I A467V 1008 V432V E437P C464I A467V 1009 V432V E437P C464I A467V 1011 E437P F442N C464I A467V 1012 E437P F442N C464I A467V 1013 E437P F442N C464I A467V 1014 E437P F442N C464I A467V 1015 E437P F442N C464I A467V 1016 E437P F442N C464I A467V 1017 E437P F442N C464I A467V 1018 E437P F442N C464I A467V 1019 E437P F442N C464I A467V 1020 E437P F442N C464I A467V 1021 E437P F442N C464I A467V 1022 E437P F442N C464I A467V 1023 E437P F442N C464I A467V 1024 E437P F442N C464I A467V 1025 E437P F442N C464I A467V 1026 E437P F442N C464I A467V 1027 E437P F442N C464I A467V 1028 E437P F442N C464I A467V 1029 E437P F442N C464I A467V 1030 E437P F442N C464I A467V 1031 E437P F442N C464I A467V 1032 E437P F442N C464I A467V 1033 E437P F442N C464I A467V 1034 V432N E437P F442N C464I A467V 1035 E437P F442N C464I A467V 1036 E437P F442N C464I A467V 1037 E437P F442N C464I A467V 1038 E437P F442N C464I A467V 1039 E437P F442N C464I A467V 1040 E437P F442N C464I A467V 1041 E437P F442N C464I A467V 1042 E437P F442N C464I A467V 1043 E437P F442N C464I A467V 1044 E437P F442N C464I A467V 1045 E437P F442N C464I A467V 1046 E437P F442N C464I A467V 1047 E437P F442N C464I A467V 1048 E437P F442N C464I A467V 1049 E437P F442N C464I A467V 1050 E437P F442N C464I A467V 1051 E437P F442N C464I A467V 1052 E437P F442N C464I A467V 1053 E437P F442N C464I A467V 1054 E437P F442N C464I A467V 1055 E437P F442N C464I A467V 1056 E437P F442N C464I A467V 1057 E437P F442N C464I A467V 1058 E437P F442N C464I A467V 1059 E437P F442N C464I A467V 1060 E437P F442N C464I A467V 1061 E437P F442N C464I A467V 1062 E437P F442N C464I A467V 1063 E437P F442N C464I A467V 1064 E437P F442N C464I A467V 1065 E437P F442N C464I A467V 1066 E437P F442N C464I A467V 1067 E437P F442N C464I A467V 1068 E437P F442N C464I A467V 1069 E437P F442N C464I A467V 1070 E437P F442N C464I A467V 1071 E437P F442N C464I A467V 1072 E437P F442N C464I A467V 1073 E437P F442N C464I A467V 1074 E437P F442N C464I A467V 1075 E437P F442N C464I A467V 1076 E437P F442N C464I A467V 1077 E437P F442N C464I A467V 1078 E437P F442N C464I A467V 1079 E437P F442N S447P C464I A467V 1080 E437P F442N S447H C464I A467V 1081 E437P F442N S447K C464I A467V 1082 E437P F442N S447R C464I A467V 1083 E437P F442N S447K C464I A467V 1084 E437P F442N S447K C464I A467V 1085 E437P F442N S447K C464I A467V 1086 E437P F442N S447R C464I A467V 1087 E437P F442N S447P C464I A467V 1088 E437P F442N S447P C464I A467V 1089 E437P F442N C464I A467V 1090 E437P F442N C464I A467V 1091 E437P F442N C464I A467V 1092 E437P F442N C464I A467V 1093 E437P F442N S447P C464I A467V 1094 E437P F442N S447T C464I A467V 1095 E437P F442N C464I A467V 1096 E437P F442N S447E C464I A467V 1097 E437P F442N S447K C464I A467V 1098 E437P F442N S447R C464I A467V 1099 E437P F442N S447P C464I A467V 1100 E437P F442N S447P C464I A467V 1101 E437P F442N S447S C464I A467V

Various activities of the ALD variants were determined and are shown in Table 2.

TABLE 2 Activities of Exemplary ALD Variants. Small Scale R-3HB In Vivo 1,3 3HBCoA/ Aldehyde/ BDO AcCoA S-3HB Specific Variant Mutations Relative to Wild-Type Ald Production¹ Specificity² Aldehyde activity³ 12 D12A, I139S, M204R, R396H yes 16 D12A, C33R, I139S, C174S, C189A, M204R, C220V, C267A, C353A, C356T, R396H, C464V 17 D12A, I139V, T143N, G167S, C174S, M204R, C220V, T230R, C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 30 E129I, C174S, C220V, C267A, C356T, R396H, * C464I, A467V 34 D12A, I139S, C174S, M204R, C220V, C267A, Yes C356T, R396H, C464I 56 D12A, I139S, C174S, M204R, C220V, C267A, yes C356T, R396H, F429Y, , E437P, F442T, C464I, A467V 71 Y107K, C174S, M204R, C220V, C267A, C356T, C464I, A467V 80 Y107K, C174S, C220V, C267A, C356T, C464I * 93 D12A, I139S, C174S, M204R, T230R, C220V, * C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 156 D12A, Y107K, C174S, M204R, C220V, C267A, * C356T, C464I, A467V 166 D12A, Y107K, C174S, C220V, C267A, C356T, * C464I 180 D12A, I139S, C174S, M204R, C220V, C267A, * C356T, R396H, C464I, A467V 182 C174S, M204R, C220V, A243P, C267A, * C356T, R396H, E437P, C464I, A467V 184 D12A, I139S, C174S, M204R, C220V, C267A, * C356T, R396H, E437P, C464I, A467V 194 I139S, C174S, M204R, C220V, C267A, C356T, * R396H, E437P, C464I, A467V 199 C174S, M204R, C220V, C267A, C356T, * R396H, F429Q, E437P, C464I, A467V 203 C174S, M204R, C220V, C267A, C356T, * R396H, F429Y, E437P, F442T, C464I, A467V 205 D12A, I139S, C174S, M204R, C220V, A243P, * C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 208 C174S, M204R, C220V, C267A, C356T, * R396H, E437P, F442Y, C464I, A467V 213 T143S, C174S, M204R, C220V, C267A, * C356T, R396H, E437P, C464I, A467V 235 D12A, I139S, C174S, M204R, C220V, A243P, * C267A, C356T, R396H, E437P, C464I, A467V 240 D12A, I139V, C174S, M204R, M227K, C220V, * C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 321 D12V, I139S, M204R, R396H * 598 D12A, I139S, C174S, M204R, M227Q, T230R, Yes + A243P, C220V, C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 45 C174S, M204R, C220V, C267A, C356T, Yes +++ + R396H, E437P, C464I, A467V 951 C174S, M204R, C220V, C267A, C356T, + + R396H, F429H, E437P, F442H, C464I, A467V 952 C174S, M204R, C220V, C267A, C356T, + R396H, F429M, E437P, F442H, C464I, A467V 953 C174S, M204R, C220V, C267A, C356T, + R396H, F429M, E437P, F442N, C464I, A467V 954 C174S, M204R, C220V, C267A, C356T, + R396H, F429Q, E437P, C464I, A467V 955 C174S, M204R, C220V, C267A, C356T, Yes +++ + R396H, E437P, F442N, C464I, A467V 957 C174S, M204R, C220V, C267A, C356T, + + R396H, E437P, F442Q, C464I, A467V 958 C174S, M204R, C220V, C267A, C356T, + + R396H, E437P, I444V, C464I, A467V 959 C174S, M204R, C220V, C267A, C356T, + + R396H, E437P, T440H, F442N, C464I, A467V 960 V19I, D122N, C174S, M204R, C220V, C267A, + C356T, R396H, F429D, E437P, F442Q, E450E, C464I, A467V 961 C174S, M204R, C220V, C267A, V315A, + + C356T, R396H, E437P, T440H, F442N, C464I, A467V 975 D12A, I139V, C174S, M204R, C220V, Yes M227Q, T230R, A243P, C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 991 D12A, I139L, T143N, C174S, M204R, C220V, T230R, A243P, C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V 992 A73S, C174S, M204R, C220V, C267A, C356T, + {grave over ( )}+ R396H, E437P, F442M, S447M, C464I, A467V 993 C174S, M204R, C220V, A254T, C267A, + C356T, R396H, E437P, F442M, C464I, A467V 994 V163C, C174S, M204R, C220V, C267A, + C356T, R396H, E437P, F442M, C464I, A467V 995 C174S, M204R, C220V, K 229S, C267A, + C356T, R396H, E437P, F442N, C464I, A467V 996 C174S, M204R, C220V, C267A, C356L, + R396H, E437P, F442N, C464I, A467V 997 C174S, M204R, C220V, C267A, C356T, + R396H, E437P, T441G, I44L, C464I, A467V 998 C174S, M204R, C220V, C267A, C356T, + {grave over ( )}+ R396H, E437P, F442M, C464I, A467V 999 K65A, C174S, M204R, C220V, C267A, C356T, + R396H, E437P, F442N, C464I, A467V 1000 V163C, C174S, M204R, C220V, C267A, + {grave over ( )}+ C356T, R396H, E437P, F442N, C464I, A467V 1001 C174S, M204R, C220V, C267A, C356T, + R396H, E437P, F442M, R460K, C464I, A467V 1002 C174S, M204R, C220V, C267A, C356T, Yes + {grave over ( )}+ R396H, E437P, F442M, S447M, C464I, A467V 1003 G155G, C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442F, C464I, A467V 1004 P145P, C174S, M204R, C220V, C267A, C356T, R396H, E437P, C464I, A467V 1005 G244G, C174S, M204R, C220V, C267A, C356T, R396H, E437P, C464I, A467V 1006 C174S, M204R, C220V, C267A, C356T, R396H, V432V, E437P, C464I, A467V 1015 C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1016 C174S, M204R, C220V, T230R, A243Q, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1017 C174S, M204R, C220V, T230H, A243Q, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1018 C174S, M204R, C220V, T230A, A243E, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1019 C174S, M204R, C220V, T230M, A243S, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1020 C174S, M204R, C220V, T230H, A243N, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1021 C174S, M204R, C220V, M227V, T230C, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1022 C174S, M204R, C220V, M227V, T230H, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1023 C174S, M204R, C220V, M227I, T230L, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1024 C174S, M204R, C220V, M227I, T230C, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1025 C174S, M204R, C220V, T230M, A243E, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1026 C174S, M204R, C220V, T230S, A243Q, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1027 C174S, M204R, C220V, M227I, T230A, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1028 C174S, M204R, C220V, T230K, C267A, C356T, yes {grave over ( )}− {grave over ( )}− R396H, E437P, F442N, C464I, A467V 1029 C174S, M204R, C220V, T230Y, A243Q, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1030 C174S, M204R, C220V, T230G, A243Q, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1031 C174S, M204R, C220V, T230M, A243K, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1032 C174S, M204R, C220V, T230T, A243L, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1033 C174S, M204R, C220V, T230I, C267A, C356T, yes {grave over ( )}++ {grave over ( )}− R396H, E437P, F442N, C464I, A467V 1034 C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, F429L, V432N, E437P, F442N, C464I, A467V 1035 C174S, M204R, C220V, T230H, C267A, yes {grave over ( )}+++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1036 C174S, M204R, C220V, T230Y, A243E, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1037 K72N, C174S, M204R, C220V, A243S, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1038 C174S, M204R, C220V, T230C, A243K, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1039 C174S, M204R, C220V, T230H, A243K, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1040 C174S, M204R, C220V, T230H, A243C, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1041 C174S, M204R, C220V, T230A, A243Q, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1042 C174S, M204R, C220V, T230S, A243C, C267A, yes {grave over ( )}+++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1043 C174S, M204R, C220V, M227V, T230S, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1044 C174S, M204R, C220V, T230H, A243M, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1045 C174S, M204R, C220V, T230A, A243K, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1046 C174S, M204R, C220V, T230W, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1047 C174S, M204R, C220V, M227C, T230R, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1048 C174S, M204R, C220V, M227L, T230N, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1049 C174S, M204R, C220V, T230N, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1050 C174S, M204R, C220V, M227C, T230L, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1051 C174S, M204R, C220V, T230V, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1052 C174S, M204R, C220V, T230L, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1053 C174S, M204R, C220V, M227C, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1054 C174S, M204R, C220V, M227C, T230V, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1055 C174S, M204R, C220V, T230T, A243N, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1056 C174S, M204R, C220V, T230T, A243I, C267A, yes {grave over ( )}+++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1057 C174S, M204R, C220V, T230T, A243C, C267A, yes {grave over ( )}+ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1058 C174S, M204R, C220V, T230G, A243K, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1059 C174S, M204R, C220V, T230R, A243K, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1060 C174S, M204R, C220V, M227L, A243P, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1061 C174S, M204R, C220V, M227A, A243P, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1062 C174S, M204R, C220V, A243Q, C267A, yes {grave over ( )}+ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1063 C174S, M204R, C220V, T230Q, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1064 C174S, M204R, C220V, T230N, A243I, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1065 C174S, M204R, C220V, T230C, A243C, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1066 C174S, M204R, C220V, M227I, T230R, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1067 C174S, M204R, C220V, M227I, A243L, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1068 C174S, M204R, C220V, M227I, A243M, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1069 C174S, M204R, C220V, A243M, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1070 C174S, M204R, C220V, M227V, C267A, yes {grave over ( )}++ {grave over ( )}− C356T, R396H, E437P, F442N, C464I, A467V 1071 C174S, M204R, C220V, M227C, A243Q, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1072 C174S, M204R, C220V, T230R, A243C, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1073 C174S, M204R, C220V, T230L, A243M, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1074 C174S, M204R, C220V, T230I, A243M, yes {grave over ( )}+++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1075 C174S, M204R, C220V, T230M, A243Q, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1076 C174S, M204R, C220V, M227L, T230W, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1077 C174S, M204R, C220V, T230V, A243M, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1078 C174S, M204R, C220V, M227V, T230I, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1079 A73D, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}++ {grave over ( )}+ C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1080 A73G, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447H, C464I, A467V 1081 A73L, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447K, C464I, A467V 1082 A73Q, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}++ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447R, C464I, A467V 1083 A73F, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447K, C464I, A467V 1084 A73G, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447K, C464I, A467V 1085 A73E, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, S447K, C464I, A467V 1086 A73W, C174S, M204R, C220V, M227I, yes {grave over ( )}++ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447R, C464I, A467V 1087 V163G, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1088 V163T, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1089 C174S, M204R, C220V, M227L, T230S, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1090 C174S, M204R, C220V, A243E, C267A, yes {grave over ( )}+ {grave over ( )}+ C356T, R396H, E437P, F442N, C464I, A467V 1091 C174S, M204R, C220V, T230T, A243E, yes {grave over ( )}+ {grave over ( )}− C267A, C356T, R396H, E437P, F442N, C464I, A467V 1092 C174S, M204R, C220V, A243K, C267A, yes {grave over ( )}+ {grave over ( )}+ C356T, R396H, E437P, F442N, C464I, A467V 1093 A73L, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}+ C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1094 A73R, C174S, M204R, C220V, M227I, T230K, yes {grave over ( )}+ {grave over ( )}+ C267A, C356T, R396H, E437P, F442N, S447T, C464I, A467V 1095 A73C, V163C, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}+ T230K, C267A, C356T, R396H, E437P, F442N, C464I, A467V 1096 V163C, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447E, C464I, A467V 1097 A73W, V163C, C174S, M204R, C220V, yes {grave over ( )}+ {grave over ( )}+ M227I, T230K, C267A, C356T, R396H, E437P, F442N, S447K, C464I, A467V 1098 A73M, V163C, C174S, M204R, C220V, yes {grave over ( )}+ {grave over ( )}+ M227I, T230K, C267A, C356T, R396H, E437P, F442N, S447R, C464I, A467V 1099 V163C, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}+ T230K, C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1100 A73F, V163C, C174S, M204R, C220V, M227I, yes {grave over ( )}+ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447P, C464I, A467V 1101 V163C, C174S, M204R, C220V, M227I, yes {grave over ( )}+++ {grave over ( )}− T230K, C267A, C356T, R396H, E437P, F442N, S447S, C464I, A467V ¹* active on other diols ²{grave over ( )}− = specificity < 1′ {grave over ( )}+ = specificity between 1.0-2.0′ {grave over ( )}++ = specificity between 2.0-3.0′ {grave over ( )}+++ = specificity > 3.0 ³{grave over ( )}− = relative activity < 1′ {grave over ( )}+ = relative activity > 1′

Additional activities of exemplary ALD variants are shown in Table 3. Levels of 1,3-BDO production at 48 hours were obtained with ALD variants as high as greater than 50 g/liter, greater than 60 g/liter, greater than 70 g/liter, greater than 80 g/liter, and greater than 90 g/liter.

TABLE 3 Activities of Exemplary ALD Variants. Stable Enzyme R-3HB Increased Increased Activity 3HBCoA/ Aldehyde/ 1,3-BDO enyzme in Crude Cofactor AcCoA S-3HB produced activity Variant Mutations Relative to Wild-Type Ald Lysates Preference Specificity Aldehyde in vivo in vitro 45 C174S, M204R, C220V, C267A, C356T, R396H, E437P, C464I, + NADH + + + A467V 951 C174S, M204R, C220V, C267A, C356T, R396H, F429H, E437P, + NADH + + + F442H, C464I, A467V 952 C174S, M204R, C220V, C267A, C356T, R396H, F429M, E437P, + NADH + + F442H, C464I, A467V 953 C174S, M204R, C220V, C267A, C356T, R396H, F429M, E437P, + NADH + + F442N, C464I, A467V 954 C174S, M204R, C220V, C267A, C356T, R396H, F429Q, E437P, + NADH + + C464I, A467V 955 C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442N, + NADH + + + C464I, A467V 957 C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442Q, + NADH + + + C464I, A467V 958 C174S, M204R, C220V, C267A, C356T, R396H, E437P, I444V, + NADH + + + C464I, A467V 959 C174S, M204R, C220V, C267A, C356T, R396H, E437P, T440H, + NADH + + + F442N, C464I, A467V 960 V19I, D122N, C174S, M204R, C220V, C267A, C356T, R396H, + NADH + + F429D, E437P, F442Q, E450E, C464I, A467V 961 C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, + NADH + + + T440H, F442N, C464I, A467V 962 A73S, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + + + F442M, S447M, C464I, A467V 963 C174S, M204R, C220V, A254T, C267A, C356T, R396H, E437P, + + + F442M, C464I, A467V 964 V163C, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + + + F442M, C464I, A467V 965 C174S, M204R, C220V, K 229S, C267A, C356T, R396H, E437P, + + + F442N, C464I, A467V 966 C174S, M204R, C220V, C267A, C356L, R396H, E437P, F442N, + + + C464I, A467V 967 C174S, M204R, C220V, C267A, C356T, R396H, E437P, T441G, + + + I44L, C464I, A467V 968 C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442M, + + + C464I, A467V 969 K65A, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + + + F442N, C464I, A467V 970 V163C, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + + + F442N, C464I, A467V 971 C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442M, + + + R460K, C464I, A467V 972 C174S, M204R, C220V, C267A, C356T, R396H, E437P, F442M, + + + S447M, C464I, A467V 598 D12A, I139S, C174S, M204R, M227Q, T230R, A243P, C220V, + NADH/ + + C267A, C356T, R396H, F429Y, F442T, E437P, C464I, A467V NADPH 973 C174S, M204R, C220V, C267A, A243K, C356T, R396H, E437P, + NADH + + F442N, C464I, A467V 974 Y107N, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + NADPH + + F442N, C464I, A467V 975 D122G, C174S, M204R, C220V, C267A, C356T, R396H, E437P, + NADPH + + F442N, C464I, A467V 976 C174S, M204R, C220V, C267A, S349T, C356T, R396H, E437P, + + + F442N, C464I, A467V 977 C174S, N201D, M204R, C220V, C267A, C356T, R396H, E437P, + + + F442N, C464I, A467V 978 C174S, M204R, C220V, C267A, D313R, C356T, R396H, E437P, NADH + + C464I, A467V 979 C174S, M204R, C220V, C267A, P348G, C356T, R396H, E437P, NADH + + C464I, A467V 980 C174S, M204R, C220V, C267A, C356L, R396H, E437P, C464I, NADH + + A467V 981 C174S, M204R, C220V, C267A, C356T, A360K, R396H, E437P, NADH + + C464I, A467V 982 C174S, M204R, C220V, A243K, C267A, C356T, R396H, E437P, NADH + + C464I, A467V 983 C174S, M204R, C220V, K258W, C267A, C356T, R396H, E437P, NADH + + C464I, A467V 984 Y107N, C174S, M204R, C220V, C267A, C356T, R396H, E437P, NADH + + C464I, A467V 985 C174S, M204R, C220V, N223Q, C267A, C356T, R396H, E437P, NADH + + C464I, A467V 986 S131A, C174S, M204R, C220V, C267A, C356T, R396H, E437P, NADH + + F442N, C464I, A467V

Such aldehyde dehydrogenase variants as described above can be used to produce a stereoisomer of R-3-hydroxybutyraldehyde or a mixture of R and S forms with a higher proportion of the R form. Such a stereoisomer can be utilized to make stereoisomers of downstream products, such as R-1,3-butanediol. Such stereoisomers have usefulness as pharmaceuticals or nutraceuticals.

These results demonstrate the production of aldehyde dehydrogenase variants having desirable properties, which are useful for commercial production of 3-hydroxybutyraldeyde, 1,3-butanediol, 4-hydroxybutyraldehyde or 1,4-butanediol or other desired products that are produced by metabolic pathways comprising an aldehyde dehydrogenase.

The variants described above are based on the ALD-1 parental sequence. It is understood that variant amino acid positions as shown in Tables 1, 2 or 3 can be applied to homologous aldehyde dehydrogenase sequences. Table 4 provides exemplary ALD sequences based on homology. One skilled in the art will readily understand that such sequences can be analyzed with routine and well known methods for aligning sequences (for example BLAST, blast.ncbi.nlm.nih.gov; Altschul et al.,” J. Mol. Biol. 215:403-410 (1990)). Furthermore, additional homologous ALD sequences can be identified by searching publicly available sequence databases such as found at the National Center for Biotechnology Information (NCBI) GenBank database, European Molecular Biology Laboratory (EMBL), ExPasy Prosite, or other publicly available sequence databases using BLAST. Such alignments can provide information on conserved residues that can be utilized to identify a consensus sequence for preserving enzyme activity as well as positions for generating further enzyme variants.

TABLE 4 Exemplary Aldehyde Dehydrogenase (ALD) Sequences. butyraldehyde dehydrogenase [Clostridium saccharoperbutylacetonicum N1-4(HMT)] AAP42563.1 GI:31075383 (SEQ ID NO: 7) hypothetical protein ROSEINA2194_01708 [Roseburia inulinivorans DSM 16841] EEG94445.1 (SEQ ID NO: 8) aldehyde dehydrogenase [Bacillus sp. FJAT-21945] KOP84001.1 (SEQ ID NO: 9) aldehyde dehydrogenase [Bacillus solani] KQL21940.1 (SEQ ID NO: 10 aldehyde dehydrogenase [Terrisporobacter othiniensis] WP_039679531.1 (SEQ ID NO: 11) aldehyde dehydrogenase [Roseburia inulinivorans DSM 16841] ABC25528.1 GI:83596371 (SEQ ID NO: 12) propionaldehyde dehydrogenase [Clostridium sp. ASF502] WP_004073235.1 (SEQ ID NO: 13) aldehyde dehydrogenase [[Bacillus] selenitireducens] WP_013174003.1 (SEQ ID NO: 14) aldehyde dehydrogenase [Blautia obeum] WP_005427729.1 (SEQ ID NO: 15) hypothetical protein CLOBOL_07248 [[Clostridium] bolteae ATCC BAA-613] EDP12494.1 (SEQ ID NO: 16) aldehyde dehydrogenase [Jeotgalibacillus alimentarius] WP_041123321.1 (SEQ ID NO: 17) aldehyde dehydrogenase (NAD) family protein [[Clostridium] hiranonis DSM 13275] EEA85935.1 (SEQ ID NO: 18) MULTISPECIES: aldehyde dehydrogenase [Thermoanaerobacter] WP_003870148.1 (SEQ ID NO: 19) MULTISPECIES: aldehyde dehydrogenase [Clostridiales] WP_008705584.1 (SEQ ID NO: 20) Aldehyde Dehydrogenase [Sebaldella termitidis ATCC 33386] ACZ07905.1 (SEQ ID NO: 21) propionaldehyde dehydrogenase [Eubacterium plexicaudatum] WP_004061597.1 (SEQ ID NO: 22) MULTISPECIES: aldehyde dehydrogenase [Escherichia] WP_000997839.1 (SEQ ID NO: 23) aldehyde dehydrogenase [Rhodospirillum rubrum] WP_011388669.1 (SEQ ID NO: 24) aldehyde dehydrogenase [Clostridium beijerinckii] WP_012060202.1 (SEQ ID NO: 25) aldehyde dehydrogenase [[Eubacterium] hallii] WP_005344386.1 (SEQ ID NO: 26) aldehyde dehydrogenase [Vibrio sp. EJY3] WP_014232054.1 (SEQ ID NO: 27) aldehyde dehydrogenase [Rhodopseudomonas palustris BisB18] ABD86737.1 (SEQ ID NO: 28) aldehyde dehydrogenase EutE [Desulfatibacillum alkenivorans] WP_015949695.1 (SEQ ID NO: 29) aldehyde dehydrogenase Ald [Clostridium saccharobutylicum] WP_022747467.1 (SEQ ID NO: 30) aldehyde dehydrogenase [Clostridium sp. DL-VIII] WP_009171375.1 (SEQ ID NO: 31) aldehyde dehydrogenase EutE [Clostridium taeniosporum] WP_069679818.1 (SEQ ID NO: 32) aldehyde dehydrogenase [Clostridium botulinum] WP_012425099.1 (SEQ ID NO: 33) aldehyde dehydrogenase [Clostridium botulinum] WP_035786720.1 (SEQ ID NO: 34) aldehyde dehydrogenase [Clostridium botulinum] WP_039308447.1 (SEQ ID NO: 35) aldehyde dehydrogenase [Clostridium botulinum] WP_035792132.1 (SEQ ID NO: 36) aldehyde dehydrogenase [Clostridium pasteurianum] WP_023973059.1 (SEQ ID NO: 37) NAD-dependent aldehyde dehydrogenase [Clostridium saccharoperbutylacetonicum] WP_015395720.1 (SEQ ID NO: 38) MULTISPECIES: aldehyde dehydrogenase [Clostridium] WP_023975647.1 (SEQ ID NO: 39) aldehyde dehydrogenase [Clostridium beijerinckii] WP_026888070.1 (SEQ ID NO: 40) Clostridium beijerinckii strain NRRL B593 hypothetical protein, coenzyme A acylating aldehyde dehydrogenase (ald), AF157306.2 acetoacetate: butyrate/acetate coenzyme A transferase (ctfA), acetoacetate: butyrate/acetate coenzyme A transferase (ctfB), and (SEQ ID NO: 41) acetoacetate decarboxylase (adc) genes (AF157306 AF132754) aldehyde dehydrogenase [Clostridium beijerinckii] WP_012059995.1 (SEQ ID NO: 42) aldehyde dehydrogenase [Clostridium beijerinckii] WP_041898834.1 (SEQ ID NO: 43) aldehyde dehydrogenase [Clostridium beijerinckii] WP_017211959.1 (SEQ ID NO: 44) aldehyde dehydrogenase EutE [Clostridium beijerinckii] WP_065419149.1 (SEQ ID NO: 45) NAD-dependent aldehyde dehydrogenase [Clostridium saccharoperbutylacetonicum N1-4(HMT)]>gb|AGF59413.1| NAD- YP_007458667.1 dependent aldehyde dehydrogenase [Clostridium saccharoperbutylacetonicum N1-4(HMT)] GI:451822466 (WP_015395720.1) (SEQ ID NO: 46) aldehyde dehydrogenase [Clostridium beijerinckii NCIMB 8052]>gb|AAQ12068.1| coenzyme A acylating aldehyde YP_001310903.1 dehydrogenase [Clostridium beijerinckii NCIMB 8052]>gb|AAQ12072.1| coenzyme A acylating aldehyde dehydrogenase GI:150018649 [Clostridium beijerinckii]>gb|AAT48939.1| aldehyde dehydrogenase [Clostridium beijerinckii]>gb|AAT66436.1| coenzyme A- (WP_012059995.1) acylating aldehyde dehydrogenase [Clostridium beijerinckii]>gb|ABR35947.1| aldehyde dehydrogenase [Clostridium beijerinckii (SEQ ID NO: 47) NCIMB 8052] coenzyme A acylating aldehyde dehydrogenase [Clostridium beijerinckii] AAD31841.1 GI:4884855 (SEQ ID NO: 48) Acetaldehyde dehydrogenase (acetylating) [Clostridium sp. DL-VIII]>gb|EHJ00721.1| Acetaldehyde dehydrogenase ZP_09206127.1 (acetylating) [Clostridium sp. DL-VIII] GI:359413662 (SEQ ID NO: 49) coenzyme A acylating aldehyde dehydrogenase [Clostridium saccharobutylicum] CAQ57983.1 GI:189310620 (SEQ ID NO: 50) ethanolamine utilization protein EutE [Clostridium botulinum B str. Eklund 17B]>gb|ACD24339.1| ethanolamine utilization YP_001886323.1 protein EutE [Clostridium botulinum B str. Eklund 17B] GI:187934965 (WP_012425099.1) (SEQ ID NO: 51) Aldehyde Dehydrogenase [Caldalkalibacillus thermarum TA2.A1]>gb|EGL82399.1| Aldehyde Dehydrogenase ZP_08533507.1 [Caldalkalibacillus thermarum TA2.A1] GI:335040377 (SEQ ID NO: 52) Aldehyde Dehydrogenase [Pelosinus fermentans DSM 17108]>ref|ZP_(——)15517111.1| Aldehyde Dehydrogenase [Pelosinus ZP_10327808.1 fermentans B4]>ref|ZP_15521980.1| Aldehyde Dehydrogenase [Pelosinus fermentans B3]>ref|ZP_15526533.1| Aldehyde GI:392962372 Dehydrogenase [Pelosinus fermentans A12]>ref|ZP_(——)15534416.1| Aldehyde Dehydrogenase [Pelosinus fermentans (SEQ ID NO: 53) A11]>gb|EIW18982.1| Aldehyde Dehydrogenase [Pelosinus fermentans B4]>gb|EIW21808.1| Aldehyde Dehydrogenase [Pelosinus fermentans A11]>gb|EIW29163.1| Aldehyde Dehydrogenase [Pelosinus fermentans DSM 17108]>gb|EIW35484.1| Aldehyde Dehydrogenase [Pelosinus fermentans B3]>gb|EIW36902.1| Aldehyde Dehydrogenase [Pelosinus fermentans A12] NAD-dependent aldehyde dehydrogenase [Thermoanaerobacterium thermosaccharolyticum M0795]>gb|AGB19701.1| NAD- YP_007299398.1 dependent aldehyde dehydrogenase [Thermoanaerobacterium thermosaccharolyticum M0795] GI:433655690 (WP_015312185.1) (SEQ ID NO: 54) Aldehyde Dehydrogenase [Pelosinus fermentans JBW45]>gb|EIW48189.1| Aldehyde Dehydrogenase [Pelosinus fermentans ZP_15537951.1 JBW45] GI:421076976 (SEQ ID NO: 55) aldehyde dehydrogenase family protein [Desulfosporosinus sp. OT]>gb|EGW35902.1| ZP_08814704.1 aldehyde dehydrogenase family protein [Desulfosporosinus sp. OT] GI:345862484 (SEQ ID NO: 56) hypothetical protein CLOSTMETH_00016 [Clostridium methylpentosum DSM 5476]>gb|EEG32278.1| hypothetical protein ZP_03705305.1 CLOSTMETH_00016 [Clostridium methylpentosum DSM 5476] GI:225016072 (SEQ ID NO: 57) aldehyde dehydrogenase [Thermoanaerobacterium saccharolyticum JW/SL-YS485]>gb|AFK85255.1| Aldehyde Dehydrogenase YP_006390854.1 [Thermoanaerobacterium saccharolyticum JW/SL-YS485] GI:390933349 (WP_014757178.1) (SEQ ID NO: 58) acetaldehyde dehydrogenase [Thermoanaerobacterium xylanolyticum LX-11]>gb|AEF18105.1| Acetaldehyde dehydrogenase YP_004471777.1 (acetylating) [Thermoanaerobacterium xylanolyticum LX-11] GI:333897903 (WP_013788835.1) (SEQ ID NO: 59) aldehyde dehydrogenase EutE [Acetonema longum DSM 6540]>gb|EGO64744.1| aldehyde dehydrogenase EutE [Acetonema ZP_08623980.1 longum DSM 6540] GI:338811775 (SEQ ID NO: 60) ethanolamine utilization protein eutE [Geobacillus thermoglucosidans TNO-09.020]>gb|EID44455.1| ethanolamine utilization ZP_17694107.1 protein eutE [Geobacillus thermoglucosidans TNO-09.020] GI:423719925 (SEQ ID NO: 61) aldehyde dehydrogenase [Geobacillus sp. Y4.1MC1]>gb|ADP74637.1| Aldehyde Dehydrogenase [Geobacillus sp. Y4.1MC1] YP_003989248.1 GI:312110932 (WP_013400810.1) (SEQ ID NO: 62) acetaldehyde dehydrogenase [Geobacillus thermoglucosidasius C56-YS93]>gb|AEH47899.1| Acetaldehyde dehydrogenase YP_004587980.1 (acetylating) [Geobacillus thermoglucosidasius C56-YS93] GI:336235364 (WP_013876899.1) (SEQ ID NO: 63) aldehyde dehydrogenase EutE [Bacillus azotoformans LMG 9581]>gb|EKN64472.1| aldehyde dehydrogenase EutE [Bacillus ZP_11313951.1 azotoformans LMG 9581] GI:410460269 (SEQ ID NO: 64) putative aldehyde dehydrogenase, ethanolamine utilization protein [[Clostridium] sticklandii]>emb|CBH20800.1| putative YP_003935705.1 aldehyde dehydrogenase, ethanolamine utilization protein [[Clostridium] sticklandii] GI:310657984 (WP_013360893.1) (SEQ ID NO: 65) Aldehyde Dehydrogenase [Thermincola potens JR]>gb|ADG81503.1| Aldehyde Dehydrogenase [Thermincola potens JR] YP_003639404.1 GI:296132157 (WP_013119524.1) (SEQ ID NO: 66) CoA-dependent propionaldehyde dehydrogenase [Clostridium sp. D5]>gb|EGB92558.1| CoA-dependent propionaldehyde ZP_08130302.1 dehydrogenase [Clostridium sp. D5] GI:325263568 (SEQ ID NO: 67) acetaldehyde dehydrogenase (acetylating) [Fusobacterium sp. 3_1_33]>gb|EEW94895.1| acetaldehyde dehydrogenase ZP_05815063.1 (acetylating) [Fusobacterium sp. 3_1_33] GI:260494934 (SEQ ID NO: 68) ethanolamine utilization protein eutE [Fusobacterium sp. 7_1]>gb|EEO43449.1| ethanolamine utilization protein eutE ZP_04573939.1 [Fusobacterium sp. 7_1] GI:237743458 (SEQ ID NO: 69) NAD-dependent aldehyde dehydrogenases [Ruminococcus sp. SR1/5]>emb|CBL20089.1| NAD-dependent aldehyde YP_007783752.1 dehydrogenases [Ruminococcus sp. SR1/5] GI:479153977 (WP_015525955.1) (SEQ ID NO: 70) hypothetical protein HMPREF9942_01197 [Fusobacterium nucleatum subsp. animalis F0419]>gb|EHO78009.1| hypothetical ZP_17125059.1 protein HMPREF9942_01197 [Fusobacterium nucleatum subsp. animalis F0419] GI:423137416 (SEQ ID NO: 71) possible aldehyde dehydrogenase [Fusobacterium nucleatum subsp. polymorphum ATCC 10953]>gb|EDK87521.1| possible ZP_04969437.1 aldehyde dehydrogenase [Fusobacterium nucleatum subsp. polymorphum ATCC 10953] GI:254302079 (SEQ ID NO: 72) ethanolamine utilization protein eutE [Fusobacterium sp. D11]>gb|EFD80567.1| ethanolamine utilization protein eutE ZP_06524378.1 [Fusobacterium sp. D11] GI:289765000 (SEQ ID NO: 73) aldehyde dehydrogenase EutE [Fusobacterium nucleatum ChDC F128]>gb|EJU08233.1| aldehyde dehydrogenase EutE ZP_15972610.1 [Fusobacterium nucleatum ChDC F128] GI:421526001 (SEQ ID NO: 74) CoA-dependent propionaldehyde dehydrogenase [Fusobacterium nucleatum subsp. polymorphum F0401]>gb|EHG19190.1| ZP_16419680.1 CoA-dependent propionaldehyde dehydrogenase [Fusobacterium nucleatum subsp. polymorphum F0401] GI:422338720 (SEQ ID NO: 75) CoA-dependent propionaldehyde dehydrogenase [Fusobacterium sp. 11_3_2]>gb|EGN65750.1| CoA-dependent ZP_08600044.1 propionaldehyde dehydrogenase [Fusobacterium sp. 11_3_2] GI:336419790 (SEQ ID NO: 76) hypothetical protein CLOSTASPAR_02210 [Clostridium asparagiforme DSM 15981]>gb|EEG55710.1| hypothetical protein ZP_03758198.1 CLOSTASPAR_02210 [Clostridium asparagiforme DSM 15981] GI:225388474 (SEQ ID NO: 77) aldehyde dehydrogenase [Clostridium phytofermentans ISDg]>gb|ABX41556.1| Aldehyde Dehydrogenase_[Clostridium YP_001558295.1 phytofermentans ISDg] GI:160879327 (WP_012199204.1) (SEQ ID NO: 78) CoA-dependent propionaldehyde dehydrogenase [Fusobacterium sp. 1_1_41FAA]>gb|EFG28139.1| CoA-dependent ZP_06748808.1 propionaldehyde dehydrogenase [Fusobacterium sp. 1_1_41FAA] GI:294783484 (SEQ ID NO: 79) hypothetical protein HMPREF0991_01940 [Lachnospiraceae bacterium 2_1_58FAA]>gbEGN47419.1| hypothetical protein ZP_08612821.1 HMPREF0991_01940 [Lachnospiraceae bacterium 2_1_58FAA] GI:336432991 (SEQ ID NO: 80) hypothetical protein RUMGNA_01022 [Ruminococcus gnavus ATCC 29149]>gb|EDN78612.1| aldehyde dehydrogenase ZP_02040258.1 (NAD) family protein [Ruminococcus gnavus ATCC 29149] GI:154503198 (SEQ ID NO: 81) NAD-dependent aldehyde dehydrogenases [Ruminococcus obeum A2-162]>emb|CBL23217.1| NAD-dependent aldehyde YP_007805199.1 dehydrogenases [Ruminococcus obeum A2-162] GI:479177598 (WP_015542038.1) (SEQ ID NO: 82) aldehyde dehydrogenase [Clostridium saccharolyticum WM1]>gb|ADL04402.1| Aldehyde Dehydrogenase [Clostridium YP_003822025.1 saccharolyticum WM1] GI:302386203 (WP_013272491.1) (SEQ ID NO: 83) aldehyde dehydrogenase family protein [Flavonifractor plautii ATCC 29863]>gb|EHM40040.1| aldehyde dehydrogenase family ZP_09385796.1 protein [Flavonifractor plautii ATCC 29863] GI:365844997 (SEQ ID NO: 84) hypothetical protein RUMOBE_00094 [Ruminococcus obeum ATCC 29174]>gb|EDM88971.1| aldehyde dehydrogenase ZP_01962381.1 (NAD) family protein [Ruminococcus obeum ATCC 29174] GI:153809713 (SEQ ID NO: 85) Aldehyde Dehydrogenase [Clostridium carboxidivorans P7]>ref|ZP_06856832.1| aldehyde dehydrogenase (NAD) family protein ZP_05391061.1 [Clostridium carboxidivorans P7]>gb|EET88516.1| Aldehyde Dehydrogenase [Clostridium carboxidivorans P7]>gb|EFG86154.1| GI:255524100 aldehyde dehydrogenase (NAD) family protein [Clostridium carboxidivorans P7]>gb|ADO12117.1| CoA- (SEQ ID NO: 86) acylating aldehyde dehydrogenase [Clostridium carboxidivorans P7] hypothetical protein FUAG_00592 [Fusobacterium ulcerans ATCC 49185]>gb|EFS25077.1| hypothetical protein FUAG_00592 ZP_10974295.1 [Fusobacterium ulcerans ATCC 49185] GI:404368948 (SEQ ID NO: 87) hypothetical protein HMPREF0402_0608 [Fusobacterium sp. 12_1B]>gb|EHO83590.1| hypothetical protein ZP_09586735.1 HMPREF0402_00608 [Fusobacterium sp. 12_1B] GI:373496187 (SEQ ID NO: 88) Aldehyde Dehydrogenase [Clostridium carboxidivorans P7]>ref|ZP_(——)06855343.1| aldehyde dehydrogenase (NAD) family protein ZP_05393779.1 [Clostridium carboxidivorans P7]>gb|EET85788.1| Aldehyde Dehydrogenase [Clostridium carboxidivorans GI:255526882 P7]>gb|EFG87815.1| aldehyde dehydrogenase (NAD) family protein [Clostridium carboxidivorans P7] (SEQ ID NO: 89) NAD-dependent aldehyde dehydrogenases [Clostridium cf. saccharolyticum K10]>emb| CBK77787.1| NAD-dependent YP_007849785.1 aldehyde dehydrogenases [Clostridium cf. saccharolyticum K10] GI:479338567 (WP_015574070.1) (SEQ ID NO: 90) ethanolamine utilization protein eutE [Fusobacterium varium ATCC 27725]>gb| EES62817.1| ethanolamine utilization protein ZP_08693593.1 eutE [Fusobacterium varium ATCC 27725] GI:340756989 (SEQ ID NO: 91) aldehyde dehydrogenase family protein [Clostridium celatum DSM 1785]>gb|EKY29259.1| aldehyde dehydrogenase family ZP_19296595.1 protein [Clostridium celatum DSM 1785] GI:429764274 (SEQ ID NO: 92) propionaldehyde dehydrogenase [Clostridium sp. ASF502] EMZ20682.1 GI:476613570 (SEQ ID NO: 93) hypothetical protein HMPREF0988_02063 [Lachnospiraceae bacterium 1_4_56FAA]>gb|EGN36620.1| hypothetical protein ZP_08616478.1 HMPREF0988_02063 [Lachnospiraceae bacterium 1_4_56FAA] GI:336436768 (SEQ ID NO: 94) hypothetical protein HMPREF0994_03038 [Lachnospiraceae bacterium 3_1_57FAA_CT1]>gb|EGN40215.1| hypothetical ZP_08607032.1 protein HMPREF0994_03038 [Lachnospiraceae bacterium 3_1_57FAA_CT1] GI:336427027 (SEQ ID NO: 95) aldehyde dehydrogenase [Ruminococcus sp. 5_1_39B_FAA]>gb|EES77009.1| aldehyde dehydrogenase [Ruminococcus sp. ZP_04856816.1 5_1_39B_FAA] GI:253579547 (SEQ ID NO: 96) CoA-dependentproprionaldehyde dehydrogenase PduP [Acetobacterium woodii DSM 1030]>gb|AFA49334.1| CoA-dependent YP_005270223.1 proprionaldehyde dehydrogenase PduP [Acetobacterium woodii DSM 1030] GI:379012411 (WP_014356934.1) (SEQ ID NO: 97) ethanolamine utilization protein EutE [Clostridium botulinum E1 str. ‘BoNT E Beluga’]>gb|EES50221.1| ethanolamine utilization ZP_04822936.1 protein EutE [Clostridium botulinum E1 str. ‘BoNT E Beluga’] GI:251780016 (SEQ ID NO: 98) ethanolamine utilization protein EutE [Clostridium botulinum B str. Eklund 17B]>gb|ACD22415.1| ethanolamine utilization YP_001885942.1 protein EutE [Clostridium botulinum B str. Eklund 17B] GI:187933041 (WP_012423269.1) (SEQ ID NO: 99) ethanolamine utilization protein EutE [Clostridium botulinum E3 str. Alaska E43] >gb|ACD53952.1| ethanolamine utilization YP_001921227.1 protein EutE [Clostridium botulinum E3 str. Alaska E43] GI:188590535 (WP_012451752.1) (SEQ ID NO: 100) propionaldehyde dehydrogenase [Eubacterium plexicaudatum ASF492] EMZ27833.1 GI:476621007 (SEQ ID NO: 101) Aldehyde Dehydrogenase [Thermosediminibacter oceani DSM 16646]>gb|ADL07333.1| Aldehyde Dehydrogenase YP_003824956.1 [Thermosediminibacter oceani DSM 16646] GI:302389135 (WP_013275382.1) (SEQ ID NO: 102) hypothetical protein HMPREF1090_01637 [Clostridium clostridioforme 90A8] ENZ17687.1 GI:480674262 (SEQ ID NO: 103) hypothetical protein HMPREF9467_03550 [Clostridium clostridioforme 2_1_49FAA]>gb|EHG29726.1| hypothetical protein ZP_09116578.1 HMPREF9467_03550 [Clostridium clostridioforme 2_1_49FAA] GI:357055510 (SEQ ID NO: 104) Aldehyde Dehydrogenase [Ilyobacter polytropus DSM 2926]>gb|ADO84118.1| Aldehyde Dehydrogenase [Ilyobacter YP_003968466.1 polytropus DSM 2926] GI:310780134 (WP_013388777.1) (SEQ ID NO: 105) hypothetical protein GCWU000342_00651 [Shuttleworthia satelles DSM 14600]>gb|EEP29295.1| hypothetical protein ZP_04454656.1 GCWU000342_00651 [Shuttleworthia satelles DSM 14600] GI:229828587 (SEQ ID NO: 106) aldehyde dehydrogenase [Clostridium beijerinckii NCIMB 8052]>gb|ABR36155.1| aldehyde dehydrogenase [Clostridium YP_001311111.1 beijerinckii NCIMB 8052] GI:150018857 (WP_012060202.1) (SEQ ID NO: 107) propionaldehyde dehydrogenase [Clostridium clostridioforme CM201]>gb|ENZ04399.1| propionaldehyde dehydrogenase ENY83847.1 [Clostridium clostridioforme 90B1]>gb|ENZ17257.1| propionaldehyde dehydrogenase [Clostridium clostridioforme GI:480639338 90A8]>gb|ENZ22132.1| propionaldehyde dehydrogenase [Clostridium clostridioforme 90A3]>gb|ENZ29200.1| (SEQ ID NO: 108) propionaldehyde dehydrogenase [Clostridium clostridioforme 90A1]>gb|ENZ64224.1| propionaldehyde dehydrogenase[Clostridium clostridioforme 90A4]>gb|ENZ70105.1| propionaldehyde dehydrogenase [Clostridium clostridioforme 90A6] aldehyde dehydrogenase (NAD) domain protein [Clostridium sp. MSTE9]>gb|EJF40077.1| aldehyde dehydrogenase (NAD) ZP_14663848.1 domain protein [Clostridium sp. MSTE9] GI:420157008 (SEQ ID NO: 109) propionaldehyde dehydrogenase [Clostridium bolteae 90B8]>gb|ENZ57487.1| propionaldehyde dehydrogenase [Clostridium ENZ31577.1 bolteae 90A5]>gb|ENZ67775.1| propionaldehyde dehydrogenase [Clostridium bolteae 90B7] GI:480688660 (SEQ ID NO: 110) hypothetical protein EUBHAL_00514 [Eubacterium hallii DSM 3353]>gb|EEG37590.1| aldehyde dehydrogenase (NAD) family ZP_03715465.1 protein [Eubacterium hallii DSM 3353] GI:225026273 (SEQ ID NO: 111) CoA-acylating propionaldehyde dehydrogenase [Halanaerobium saccharolyticum subsp. saccharolyticum DSM ZP_23773859.1 6643]>emb|CCU77919.1| CoA-acylating propionaldehyde dehydrogenase [Halanaerobium saccharolyticum subsp. GI:470960332 saccharolyticum DSM 6643] (SEQ ID NO: 112) hypothetical protein [Eubacterium limosum KIST612]>gb|ADO39014.1| hypothetical protein ELI_4072 [Eubacterium limosum YP_003961977.1 KIST612] GI:310829620 (WP_013382321.1) (SEQ ID NO: 113) aldehyde dehydrogenase [Thermoanaerobacter sp. X514]>ref|ZP_07131928.1| Aldehyde Dehydrogenase [Thermoanaerobacter YP_001663556.1 sp. X561]>ref|YP_003903905.1| aldehyde dehydrogenase [Thermoanaerobacter sp. X513]>ref|ZP_08212082.1| Aldehyde GI:167040571 Dehydrogenase [Thermoanaerobacter ethanolicus JW 200]>gb|ABY93220.1| aldehyde dehydrogenase [Thermoanaerobacter sp. (WP_003870148.1) X514]>gb|EFK84693.1| Aldehyde Dehydrogenase [Thermoanaerobacter sp. X561]>gb|ADN54614.1| Aldehyde (SEQ ID NO: 114) Dehydrogenase [Thermoanaerobacter sp. X513]>gb|EGD51928.1| Aldehyde Dehydrogenase [Thermoanaerobacter ethanolicus JW200] aldehyde dehydrogenase [Rhodospirillum rubrum ATCC 11170]>ref|YP_006047210.1| aldehyde dehydrogenase EutE YP_426002.1 [Rhodospirillum rubrum F11]>gb|ABC21715.1| Aldehyde dehydrogenase [Rhodospirillum rubrum ATCC GI:83592250 11170]>gb|AEO47413.1| aldehyde dehydrogenase EutE [Rhodospirillum rubrum F11] (SEQ ID NO: 115) CoA-dependent propionaldehyde dehydrogenase [Eubacterium yurii subsp. margaretiae ATCC 43715]>gb|EFM39950.1| CoA- ZP_07453625.1 dependent propionaldehyde dehydrogenase [Eubacterium yurii subsp. margaretiae ATCC 43715] GI:306819974 (SEQ ID NO: 116) aldehyde dehydrogenase (NAD) domain protein [Eubacterium sp. AS15]>gb|EJP26117.1| aldehyde dehydrogenase (NAD) ZP_10828060.1 domain protein [Eubacterium sp. AS15] GI:402309064 (SEQ ID NO: 117) aldehyde dehydrogenase EutE [Vibrio sp. EJY3]>gb|AEX22176.1| aldehyde dehydrogenase EutE [Vibrio sp. EJY3] YP_005023154.1 GI:375265711 (WP_014232054.1) (SEQ ID NO: 118) hypothetical protein HMPREF9629_00032 [Eubacteriaceae bacterium ACC19a]>gb|EHL16790.1| hypothetical protein ZP_09320518.1 HMPREF9629_00032 [Eubacteriaceae bacterium ACC19a] GI:363893420 (SEQ ID NO: 119) aldehyde-alcohol dehydrogenase domain protein [Propionibacterium propionicum F0230a]>gb|AFN47240.1| aldehyde-alcohol YP_006513121.1 dehydrogenase domain protein [Propionibacterium propionicum F0230a] GI:397671586 (WP_014847902.1) (SEQ ID NO: 120) hypothetical protein HMPREF9628_01348 [Eubacteriaceae bacterium CM5]>gb|EHL19659.1| hypothetical protein ZP_09316712.1 HMPREF9628_01348 [Eubacteriaceae bacterium CM5] GI:363889349 (SEQ ID NO: 121) aldehyde dehydrogenase (NAD) family protein [Eubacteriaceae bacterium OBRC8]>gb|EJU23517.1| aldehyde dehydrogenase ZP_10886417.1 (NAD) family protein [Eubacteriaceae bacterium OBRC8] GI:402837902 (SEQ ID NO: 122) aldehyde dehydrogenase [Clostridium beijerinckii] AAT48939.1 (SEQ ID NO: 123)

It is understood that the individual ALD variants such as those described above can be used alone, or can be combined with any other variant amino acid position, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, that is, up to all variant amino acid positions as disclosed herein (see Tables 1-3), to generate additional variants having desirable activities. Exemplary ALD variants include, but are not limited to, single substitutions, or a combination of one or more of the substitutions, at amino acid positions disclosed in any of Tables 1-3, for example, at amino acid position 12, 19, 33, 44, 65, 72, 73, 107, 122, 129, 139, 143, 145, 155, 163, 167, 174, 189, 204, 220, 227, 229, 230, 243, 244, 254, 267, 315, 353, 356, 396, 429, 432, 437, 440, 441, 442, 444, 447, 450, 460, 464, or 467 corresponding to the amino acid sequence of ALD-1 (SEQ ID NO:1) (see Tables 1-3). For example, the ALD variants include, but are not limited to amino acid substitution, single substitutions, or a combination of one or more of the substitutions, at amino acid positions D12, V19, C33, I44, K65, K72, A73, Y107, D122, E129, I139, T143, P145, G155, V163, G167, C174, C189, M204, C220, M227, K229, T230, A243, G244, A254, C267, V315, C353, C356, R396, F429, V432, E437, T440, T441, F442, I444, S447, E450, R460, C464, or A467 corresponding to the amino acid sequence of ALD-1 (SEQ ID NO:1) (see Tables 1-3). It is understood that any substitution of the other 19 amino acids can be done at one or more desired amino acid positions.

In one embodiment, the variant ALD comprises an amino acid substitution at position 12 that is D12A. In one embodiment, the variant ALD comprises an amino acid substitution at position 19 that is V19I. In one embodiment, the variant ALD comprises an amino acid substitution at position 33 that is C33R. In one embodiment, the variant ALD comprises an amino acid substitution at position 44 that is I44L. In one embodiment, the variant ALD comprises an amino acid substitution at position 65 that is K65A. In one embodiment, the variant ALD comprises an amino acid substitution at position 72 that is K72N. In one embodiment, the variant ALD comprises an amino acid substitution at position 73 selected from A73S, A73D, A73G, A73L, A73Q, A73F, A73E, A73W, A73R, A73C, and A73M. In one embodiment, the variant ALD comprises an amino acid substitution at position 107 that is Y107K. In one embodiment, the variant ALD comprises an amino acid substitution at position 122 that is D122N. In one embodiment, the variant ALD comprises an amino acid substitution at position 129 that is E1291. In one embodiment, the variant ALD comprises an amino acid substitution at position 139 selected from I139S, I139V, and I139L. In one embodiment, the variant ALD comprises an amino acid substitution at position 143 that is T143N or T143S. In one embodiment, the variant ALD comprises an amino acid substitution at position 163 selected from V163C, V163G and V163T. In one embodiment, the variant ALD comprises an amino acid substitution at position 167 that is G167S. In one embodiment, the variant ALD comprises an amino acid substitution at position 174 that is C174S. In one embodiment, the variant ALD comprises an amino acid substitution at position 189 that is C189A. In one embodiment, the variant ALD comprises an amino acid substitution at position 204 that is M204R. In one embodiment, the variant ALD comprises an amino acid substitution at position 220 that is C220V. In one embodiment, the variant ALD comprises an amino acid substitution at position 227 selected from M227K, M227Q, M2271, M227V, M227C, M227L, and M227A. In one embodiment, the variant ALD comprises an amino acid substitution at position 229 that is K 229S. In one embodiment, the variant ALD comprises an amino acid substitution at position 230 selected from T230R, T230K, T230H, T230A, T230M, T230C, T230L, T230S, T230Y, T230G, T230T, T230I, T230W, T230N, T230V, and T230Q. In one embodiment, the variant ALD comprises an amino acid substitution at position 243 selected from A243P, A243Q, A243E, A243S, A243N, A243K, A243L, A243C, A243M, and A243I. In one embodiment, the variant ALD comprises an amino acid substitution at position 254 that is A254T. In one embodiment, the variant ALD comprises an amino acid substitution at position 267 that is C267A. In one embodiment, the variant ALD comprises an amino acid substitution at position 315 that is V315A. In one embodiment, the variant ALD comprises an amino acid substitution at position 353 that is C353A. In one embodiment, the variant ALD comprises an amino acid substitution at position 356 that is C356T or C356L. In one embodiment, the variant ALD comprises an amino acid substitution at position 396 that is R396H. In one embodiment, the variant ALD comprises an amino acid substitution at position 429 selected from F429Y, F429Q, F429H, F429M, F429D, and F429L. In one embodiment, the variant ALD comprises an amino acid substitution at position 432 that is V432V or V432N. In one embodiment, the variant ALD comprises an amino acid substitution at position 437 that is E437P. In one embodiment, the variant ALD comprises an amino acid substitution at position 440 that is T440H. In one embodiment, the variant ALD comprises an amino acid substitution at position 441 that is T441G. In one embodiment, the variant ALD comprises an amino acid substitution at position 442 selected from F442T, F442Y, F442H, F442N, F442Q, F442M, and F442F. In one embodiment, the variant ALD comprises an amino acid substitution at position 444 that is I444V. In one embodiment, the variant ALD comprises an amino acid substitution at position 447 selected from S447M, S447P, S447H, S447K, S447R, S447T, S447E, and S447S. In one embodiment, the variant ALD comprises an amino acid substitution at position 460 that is R460K. In one embodiment, the variant ALD comprises an amino acid substitution at position 464 that is C464V or C464I. In one embodiment, the variant ALD comprises an amino acid substitution at position 467 that is A467V. Any of the above-described amino acid positions can be used for single amino acid substitutions, or a combination of one or more of the substitutions, to generate an ALD variant of the invention.

Based on the teachings herein, a person skilled in the art can readily identify amino acid positions corresponding to any of amino acid positions 12, 19, 33, 44, 65, 72, 73, 107, 122, 129, 139, 143, 145, 155, 163, 167, 174, 189, 204, 220, 227, 229, 230, 243, 244, 254, 267, 315, 353, 356, 396, 429, 432, 437, 440, 441, 442, 444, 447, 450, 460, 464, or 467 corresponding to the amino acid sequence of ALD-1 (SEQ ID NO:1) in homologous ALD sequences. For example, as shown in the alignment in FIG. 4A, amino acid 1139 of ALD-1 corresponds to amino acid 1133 of SEQ ID NO:13 and 20. For SEQ ID NO:24, the corresponding position is V199. Using well known methods for aligning amino acid sequences, generally using default parameters as disclosed herein, a person skilled in the art can readily determine an amino acid position in another ALD sequence that corresponds to any of amino acid positions 12, 19, 33, 44, 65, 72, 73, 107, 122, 129, 139, 143, 145, 155, 163, 167,174,189,204,220,227,229,230,243,244,254,267,315,353,356, 396,429,432,437, 440, 441, 442, 444, 447, 450, 460, 464, or 467 corresponding to the amino acid sequence of ALD-1 (SEQ ID NO:1).

It is further understood that an ALD variant can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, that is, up to all variant amino acid positions as disclosed herein, for example, in Tables 1-3. A person skilled in the art can readily generate an ALD variant based on any single or combination of amino acid substitutions, as disclosed herein, such as the amino acid variant positions described above and in Tables 1-3. In a particular embodiment, the ALD variants are those disclosed in Tables 1-3.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank accession version designations and/or GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. 

1. An isolated nucleic acid molecule selected from: (a) a nucleic acid molecule encoding an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or 7-123, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123; (b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes the one or more amino acid substitutions; (c) a nucleic acid molecule encoding an amino acid sequence comprising the consensus sequence of Loop A (SEQ ID NO:5) and/or Loop B (SEQ ID NO:6), wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123; and (d) a nucleic acid molecule that is complementary to (a) or (b).
 2. The isolated nucleic acid molecule of claim 1, wherein said amino acid sequence, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced in SEQ ID NO: 1, 2 or 3 or 7-123.
 3. The nucleic acid of claim 1, wherein the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acid substitutions.
 4. A vector containing the nucleic acid molecule of claim
 1. 5-6. (canceled)
 7. An isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or in Table 4, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123.
 8. (canceled)
 9. An isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or in Table 4, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123, wherein said amino acid sequence, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acids sequence referenced as SEQ ID NO: 1, 2 or 3 or 7-123.
 10. The isolated polypeptide of claim 9, wherein said amino acid sequence further comprises a conservative amino acid substitution at from 1 to 100 amino acid positions, wherein said positions are other than the one or more amino acid substitutions.
 11. The isolated polypeptide of claim 7, wherein said amino acid sequence comprises no modification from 2 to 300 amino acid positions compared to the parent sequence, other than the one or more amino acid substitutions.
 12. The isolated polypeptide of claim 7, wherein the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acid substitutions set forth. 13-23. (canceled)
 24. A cell comprising the nucleic acid of claim
 1. 25-40. (canceled)
 41. A composition comprising the polypeptide of claim 7 and at least one substrate for said polypeptide. 42-25. (canceled)
 46. Culture medium comprising the cell of claim
 24. 47. (canceled)
 48. A method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, or 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing said cell of claim 24 to produce 3-HBal and/or 1,3-BDO, or an ester or amide thereof. 49-57. (canceled)
 58. 3-Hydroxybutyraldeyde (3-HBal) and/or 1,3-butanediol (1,3-BDO) or the 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO) having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, wherein the 3-HBal and/or 1,3-BDO or the 4-HBal and/or 1,4-BDO is produced by the method of claim 48, wherein the 3-HBal and/or 1,3-BDO or the 4-HBal and/or 1,4-BDO is enantiomerically enriched for the R form. 59-63. (canceled)
 64. A composition comprising the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, of claim 58, or a cell lysate or culture supernatant of a cell producing the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO.
 65. A product comprising the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, of claim 58, wherein said product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-hydroxybutyrate (P4HB) or a co-polymer thereof, poly(tetramethylene ether) glycol (PTMEG), polybutylene terephthalate (PBT), polyurethane-polyurea copolymer, nylon, organic solvent, polyurethane resin, polyester resin, hypoglycaemic agent, butadiene or butadiene-based product. 66-69. (canceled)
 70. A process for producing the product of claim 65, comprising chemically reacting the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, with itself or another compound in a reaction that produces said product.
 71. (canceled)
 72. A method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising providing a substrate to the polypeptide of claim 7 and converting the substrate to 3-HBal and/or 1,3-BDO, wherein the substrate is a racemic mixture of 1,3-hydroxybutyryl-CoA.
 73. (canceled)
 74. A method for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising providing a substrate to the polypeptide of claim 7 and converting the substrate to 4-HBal and/or 1,4-BDO, wherein the substrate is 1,4-hydroxybutyryl-CoA.
 75. (canceled)
 76. A method for producing 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO, comprising incubating a lysate of the cell of claim 24 to produce 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO. 77-79. (canceled) 